What is the best tweak?

Assuming you have all of the equipment you want (for now at least). What is the single best tweak for a system?

Speaker Cables
Power Cords
Isolation Devices
Room Acoustics
There is Not Any 1 BEST Tweak for ALL Systems - I feel!
Tweaking, as the word might describe, is Trying different things ( comparisons ) to see what Works/Sounds the best.
I have had Good Results with Some of Them and Not with All of them.
Its the 'SUM OF ALL THE PARTS' for a great sounding system!
I would rate power cords and isolation as the first areas to concentrate on. By searching for bargains on used stuff, you can economically address all these issues in increments, and realize the benefits for yourself.
After room acoustics, it's probably dedicated lines.
I gotst' all the equipment I need right here my friend. My favorite tweak is puttin' that equipment to some good use. Nothing like the nasty to put me in the mood for listening. Everything sounds better after dockin' your Johnson in the tunnel of love. I guess that falls under "other".

Other than that "other", the Room Acoustics/treatment is going to likely have the potential for a far greater effect than any other tweak you mention, unless you happen ot have really bad power in your area, in which case conditioning may have a significant effect as well. Not that the other things are not capable of making a difference. I just think that difference will be smaller and less significant. IMO of course, the room rules!

Room acoustics is very important and can be very expensive (is that a tweak?). I've found that speaker cables are critical to the sound of a system so that would be #2 (could also be pretty expensive). For lower cost tweaks, isolation devices on cd players do make a significant difference.
Room acoustic by far. The biggest difference in sound will come from this area whether good or bad. If you have "good" sounding equipment, tweaking is just what the word means. You are squeezing the best performance of what you already have. Whether that performance is good or bad is up to your ears to decide.
Since your hearing is the most important aspect of hearing sound, I'd suggest the best tweak is having your ears cleaned of any wax!
Careful speaker/listener positioning and similar setup issues are critical. They also cost next to nothing.
Each system is different, for example my CDP isolation device works great on my Sony but does nothing for my friends tubed Metronome CDP. My friedns Elrods made a huge improvement in his system but only a nice change in sound in mine. My biggest improvement was speaker spikes with the B&Ws but no so much with the Talons.

The basic answer is that you have to hear them in your system to see what works for you.

Happy Listening.
Ear wax removal. Seriously.
Better and cheaper than most anything else.
Clean and treat ALL of your connections.

Power cords: Unplug from wall. Use a small fine toothed metal flat-file to file-flat both plug flanges (4 sides). Most plug flanges I've seen are stamped (by some kind of punch-press) and have a fine ridge, along the edge, that restricts contact with the female receptical (Seinfeld fans: "the holes").
File down until no more ridges and the blade surfaces are shiny and even.
Note: Some flanges have a raised bump running down the middle. DO NOT attempt to file the whole dang thing flat! Use common sense!
Next, use "Tarn-X" the deoxify the copper/brass plug flanges. This stuff can be found in most large super markets and makes metal look like new.
Finally, plug-and-unplug about 6 times to scrape off any oxidation from the female receptical. This is the best you can do until someone invents a $100 receptical that can be disassembled for cleaning (you can't expect this for a mere $50 dollars).

Speaker cable and ICs: Unplug and clean with alcohol first (the "non-sticky when dry" kind). Use your flat file on spade lugs where appropriate (forget it with those fancy Kimber kind). Again, you're trying to make the spade surface as flat as possible for maximum contact. use the file on amp connections and speaker terminals where appropriate (I don't have to tell you to unplug the amp first, do I?). Use your Tarn-X here too if connections are copper.
After filing and cleaning, apply something like "Pro Gold" or "Tweek" (follow directions) for maximum contact. For female RCA jacks (Jills?), pull most of the cotton from an ear swab and roll remaining cotton TIGHT! Dunk it in the non-sticky alcohol and "do" the inside of the jack.
Wait a while until all alcohol evaporates.
After all of this work (takes 2-3 hours) you'll be so tired that anything you play will sound fabulous!
Isolate your power cords. Be certain they are not touching any interconnect or speaker cable. Thats number 1 because until you do that, you cannot truly judge anything else.
Got to agree with Onhwy61. Positioning is number one. Consider all the angles of your speaker orientation, do they aim up down or straight ahead, toed in or straight, different heights if possible. Try them farther from and closer to the wall than you might think, especially if you have a sub. Position yourself, be aware of reflections from sides, back and coffee table especially where the sound travel path will have a difference of less than 5 ft. Try the differend phase switch positions on your sub. Then, in two months or so, you can worry about the things that cost money.
Dweller- you should also use you cleaning treatment on the main ground connection to your home. Clean and polish the ground wire, clamp and ground rod.

I use a high speed 3M buffing wheel mounted on a bench grinder to deal with AC connectors and spade lugs.
Kana813: The buffer will make the plug flanges look better but the flat file will make them uniformly flat for more contact. I've filed down many plugs that looked flat but had tiny hills and ridges on them. Good idea about the grounding rod. How often is this necessary?
Dweller- My home is 15 years old, and this year was the first time I cleaned the grounding contacts. I guess it depends on where you live and the water/salt content in the air. I treated contact area with Caig R5 Power Booster(try some of this on your plugs), so maybe I'm good for another 15 years.

PS- before you disconnect the ground clamp for cleaning, run a jumper/bypass connection, just to be on the safe side.
Each one of your listed tweaks has had a major effect on my system. My Golden tympanics think that they hear a difference with the Cryoed Hubbell outlets. That may be the power of suggestion, for me, but the placebic effect (having a wife, who is a pharmaceutical rep, helps) may be what I'm really hearing. That little tweak, as well as cleaning posts etc. are an accumulative thing. I'm a nut job with electricity tweaking. Electrical properties and how they affect sound are quite a mysterious thing. I am going on my third power conditioner. I am waiting on delivery of my new Audio Magic Eclipse. My Stealth, and then my Matrix have been wonderful additions. In fact each one of those things on your list had major influences on my system. Perhaps, because I'm waiting on this new electrical wonder, that's what's on my mind, most. After break in, stay tuned for my thoughts. I, as well as all of the audiophools out there, can ramble on (quite cogently) about any one of those things on your list. I don't think of them as tweaks. They are major components of a sound system. Cleaning posts? Cryoed outlets? Spades rather than banana clips? Those are tweaks. peace, warren
Viva la differance, I now own 3, two that work and one for parts just in case one breaks.

If you own vinyl and can find one, buy it.

Here's a general answer to a general question. Pull your speakers at least three feet from their rear wall and put bookshelves(with books) or a cloth curtain behind them. Put large potted plants with small leaves at the first reflection points and a tapestry behind your listening couch.
What is the best part of mathematic to know?
Alternative: skip all $$$ tweaks: No $1,000. cables, no $700 powercords, No $300 jars of rocks... no $200 cones...
Save all that money, and just get better primary equipment in the first place.
With better equipment, you can skip the super tweaks... in fact you can use cheaper interconnects and power cords, and STILL HAVE BETTER SOUND, than with lesser equipment, and all the tweaks in the world.
Yes I am saying, for the money spent, a better grade of basic gear is worth WAY more, than a lesser grade of gear with money thrown at a pile of tweaks.
My (humble) $0.02/experience from 38 years of audio insanity
Geometry..No such thing as isolation ..Dampening sucks the life out of the music..Geometry redirects the musical performance to the listener and can redirect resonant energy to ground. Preserve the performance..Long live the music..Tom
Tbg...how to spel it. ;)
Ditto to Elizabeth's idea......Many paths to salvation fortunately.....Peace,Bob
First best tweak is washing all your vinyl with a good quality cleaning system. Many excellent systems exist. For the budget minded, the Disc Doctor system with manually operated vacuum machine is outstanding. You won't beleive your ears!
Second is running your entire system on its own dedicated electrical line from the breaker box. From a 20 amp breaker run 10/2 Romex up to two high quality 'hospital grade' outlets. Your components will appreciate their own food source and reward you with improved sound!
DIY:tube traps,acoustic panels,RPG skyline diffusers.They were the icing on the cake.
Power cables. Never thought a ac cable would have as much impact as compared to isolation, speaker cables or anyother tweak. The ironic thing is the power cables were the least expensive of everything else. Go figure.