Interesting.
I guess the problem that I see is that very few villains who haven't been used before seem up to the task of following Joker and Two-Face as top credible threats and interesting characters.
I mean, who do we have?
Black Mask
The Ventriloquist
Killer Croc
Deadshot
Clayface
Firefly
Hush
Mad Hatter
Hugo Strange...
What would you go with?
I think what it all comes down to is characterization. If even a seemingly weaker villain is used but depicted in a menacing enough way, he would be great. That said, I can't see Nolan using Clayface (although a certain depiction of Basil Karlo might possibly work), Manbat, Mionk (because of the Vampire thing), Mr. Freeze, Blockbuster, Dr. Phosporus (although I would love to see him in a film) or even Killer Croc or Bane(unless the venom thing was minimized or taken out but that is a huge part of who he is). Nolan also said seperately somewhere that Penguin is "not realistic enough" so even if he does potentially use Catwoman, he is unlikely to use Penguin. So really the options are:
1. Riddler
2. Catwoman
3. A return of Two-Face (depending on how exactly they treat him at the end of TDK... many say he won't be back but it is at least theoretically possible even if unlikely)
then you have a bunch of villains that could potentially be "realistic" enough and a threat if depicted in a good enough light:
The Wrath (re-written as another man who dresses up like Batman and goes around killing the few good cops (other than Gordon)... media begins blaming Batman... Bruce comes back and there is a big fight between the batmen.... the real Batman wins and redeems himself but most people still don't think he actually exists)
Black Mask
Scarface
Deadshot
Firefly
Hugo Strange
Mad Hatter
Doctor Death
Anarky
Lady Shiva (but might be likened too much to Elektra from the Daredevil movies)
Talia (if not exactly depicted as she is in the comics (more of a villain than just trying to help her father))
Cornelius Stirk (probably a secondary character)
Film Freak (as a serial killer obsessed with recreating movie scenes; secondary character)
Calendar Man (as a killer who only acts on certain days, probably as a secondary character)
Cluemaster (but would be pretty lame regardless of what Nolan might do to him as a secondary character)
Ratcatcher (secondary character)
Maxie Zeus (if toned down a little, as a secondary character)
Harley Quinn (unlikely though given that the Joker probably won't be back)
Maybe even Poison Ivy (if a severe re-write is done so she is mostly just a whacked out environmentalist-terrorist who use plant toxins etc.)
just because a character is weaker in the comics doesn't necessarily mean Nolan can't depict them as a major threat if he plays around with their characterization and maybe their story a little
Personally, would hate to see Hush... way overrated character