So I guess this is the topic for roster speculation. I looked around before making a topic of my own, so I'm bumping this one up instead. Although I did learn through that search that @MindChamber is looking into making a sequel (needs a team, though), so I guess this topic has changed from hypothetical speculation to just speculation.
One thing we might want to keep in mind is that while some characters might be added, others might be removed because they were big back in the early 2000s, and aren't as much anymore. Like, when's the last time anyone has ever brought up Samurai Asshole, for example? I think that's a pretty safe cut. Also, gee, Pico, how come daddy Fulp let your series have 4 fighters when every other series only got one? It was fine before, but there are a lot more icons on Newgrounds today that would like those roster slots.
Also, where do we stand on games that started life on Newgrounds, but went on to become bigger projects that are sold in stores, like Super Meat Boy, Castle Crashers, or The Binding of Issac? Seeing as Hank and Alien Hominid are already in the first game, I think characters from these bigger games might be ok. I don't think every game that has a demo on Newgrounds would work, though. Like, CrossCode has a demo on Newgrounds, but would you really call it a Newgrounds game? (Not that I wouldn't love to see Lea here, I just think it's not happening)
With that in mind, here's my wild roster prediction:
Cuts: Samurai Asshole, Salad Fingers, Nene, Piconjo
Additions: Meat Boy, Black Knight (Castle Crashers), Issac, Boyfriend, Tricky, Ritz the Rat, Matt (Epic Battle Fantasy), Witch (Dead Estate)
I'm not versed enough on Newgrounds history to know who started here or on a different site, aside from the most obvious picks that everyone else has pointed out already, so my picks were pretty safe. This is just some ideas to get the ball rolling on this topic again.