The Gabriel Knight adventure games are one of the earliest examples, with Tim "Wasn't cast as The Jokerbecause his audition was too frightening" Curry as The Hero and Mark Hamill as his bumbling sidekick.
Not to mention Deadpool in Marvel vs. Capcom 3, one of his highest-pitched and least serious roles he's ever had.
He reprises his role of Deadpool in his own self-titled video game, albeit with a somewhat deeper tone in general. He also does the voices in Deadpool's head, one speaking in an even higher pitch, and another deeper voice with a faux-British accent.
David in The Last of Us is probably one of the biggest examples. What makes this so shocking is that when you meet him, he actually seems like a kind gent who has people's best interests in heart... Then comes the fact that he's the leader of a bunch of raiders you killed earlier in the game. Oh, he and his boys happen to be cannibals. And David is also implied to be a pedophile. Hell, people didn't even know that Nolan even voiced him till the credits, practically like Kevin Spacey in Se7en.
Rob Paulsen as Gray Fox in the remake Metal Gear Solid: The Twin Snakes. Basically, the fact that Rob Paulsen played a very serious character (a character who is implied to be driven insane from experimentation as well as at times remorseful of his past in war, and in one source was implied to have PTSD) and do so in-character is a very sharp turn against some of his more lighthearted and comical roles.
This was done with Naked Snake in Metal Gear Solid 3: Snake Eater. Motosada Mori, a martial arts expert who was hired to do the motion capture for Snake's fight scenes, was also made to do all the intimate scenes with Eve. This led to the unintentional but absolutely Adorkable flinch away from her when she tried to kiss him, among other memorable scenes.
From Dragon Age: Origins, the first game, we have Tim Curry as Arl Rendon Howe. While Howe is a villain, Curry is usually known for playing the likable Evil Is Cool and Evil Is Hammy type villains. Arl Howe, on the other hand, is incredibly subtle and downplayed and it ends up with Howe being one of the most despicable villains he's ever played.
Charlie Adler plays Super-Skrull in Marvel vs. Capcom 3. While he's played Super-Skrull in some of the villain's animated appearances, he's way better known for playing small Funny Animal characters than intergalactic warlords.