Definite bat-spoilage ahead:

So far we've had Batman himself (Kevin Conroy) slip that Two-Face will be in the game (as we also got hints from the teaser). Here's an excerpt from 2010 Chicago C&E that was covered by comicbookresources.com:

Another fan tricked Conroy into revealing that Two-Face was a character in the upcoming "Batman: Arkham Asylum" sequel, causing a moment of silence from the actor while the audience laughed and cheered. "There's a very complicated cast," he laughed after collecting himself. "A lot of villains are in it. I'll just leave it at that!"

So what other famous villains will make their Arkham debut this turn around? Maurice LaMarche (whom voice acts for various cartoons) will be voicing Mr. Freeze and Stana Katic (from TV's Castle) will be voicing Talia al Ghul (the daughter of Ra's al Ghul and one of Batman's complicated love interests from the comics).

Plus it's a pretty safe bet we'll be seeing Joker and Harley again, so there's 5 so far, a very promising start. However, there was also that hint to The Penguin in the teaser, so perhaps 6?

Stay tuned!