A case of rather hilarious Gameplay and Story Segregation; when you go to Doom's Castle in the first game, you end up fighting a bunch of evil clones of heroes, and a few heroes who were just mind controlled. One such mind controlled hero is Colossus...except Colossus is one of the playable heroes. This gets even better if Storm and Colossus are on the team at the same time; since you're fighting Dark Captain America and Dark Colossus, there will be a special conversation where Storm points out that Captain America is certainly a dark clone (probably due to the fact Captain America is one of the poster boys of the game, meaning they couldn't get away with Dark Captain America actually being Captain America) and then explicitly states that Dark Colossus is the original Colossus... right in front of Colossus.Mind Screw doesn't even begin to explain this.
Almost anything involving Deadpool. A couple good examples:
The entirety of the conversation with Weasel, should Deadpool be with you.
Deadpool: WEASEL! Old buddy... Haven't seen you in weeks. Where've ya been hiding?
Weasel: In the hospital... You stabbed me in the leg, remember?
Deadpool: Oh yeah, that's right. But I had to. You were trying to eat the last cheesy puff.
Weasel: Ah... It... It was my bag of cheesy puffs!
Deadpool: Well, that's not how I remember it!
Deadpool's simulation mission in the original has him claiming his origin is a mixture of Storm, the Hulk, and Thor's, and then further going on to infuriate Arcade by enjoying being in Murderworld. He even mentions in the main game that the only letdown of being there was that he did not get a T-shirt (while talking to Vision in the Sanctum Sanctorum).
Deadpool arguing with the developers in the first game's credits sequence.
Deadpool: I can take down any of those goody-two-shoes wimps!
Designer: The Hulk?
Deadpool: Okay, maybe not him, bu-
Designer: The Thing?
Deadpool: Sure! If he had a cold....
Deadpool: Look Poindexter...
Deadpool's unique conversations in MUA1 especially his talk with the Ancient One.
"Stretchy man suggest Hulk release anger with words. Hulk call it 'Hulk-ku'"
"Hulk smash wide load sign;"
"Not appreciate insult;"
"Hurt Hulk's self image."
"Hulk break lots of things
"Everything make noise when broke"
"Hank wrong about mimes."
Or the best:
"Hulk want hug kitties;"
"But they so easy to squish;"
"Hulk live in cruel world."
The recording Fury left for Stark:
Computer: (Fury is trying to activate the self-destruct protocol for Prison 42) There is not enough free memory to run this program. Quit one or more programs, and try again.
Fury: Stark... your IT department sucks.
Some of the early boss battles can be this when it's 4 on 1 against a fairly easy foe. Bullseye in particular will end up getting curbstomped by four heroes in a pretty hilarious image.
An announcement comes over the PA in Stark Tower saying "Warning, a small thermonuclear device has gone missing, and Deadpool was last seen in the weapons lab". Later on, you can hear one announce that Deadpool has returned the device - it didn't make a very good basketball.
The Stark Tower PA is good for a lot of funny, from Johnny Storm leaving a prank "Happy 65th birthday, Ben Grimm" announcement to reminders that "Miss Bambi and Miss Heather" are still waiting for Tony in the main lobby.
Deadpool will make an interesting comment if you have him in your party when you go anti-registration.
Commander Maria Hill: You don't know what you're doing!
Deadpool: Par for the course, crazy lady! Besides, I can always go the other way on my next play-through.
Dr. Doom (even if beating a basic mook): *levels up* Finally! I have achieved my ultimate triumph!
During the first intro movie in MUA 1, Wolverine is seen pulling a large piece of shrapnel from his back while Thor, Captain America and Spider-Man look on.
Wolverine: What are ya girls lookin' at?
The credits of the first game has several of the characters recording their lines. They're guaranteed to make you laugh.
Xavier gets into the mind of the script writer to stop making him repeat his lines or else he'll make him relieve a very bad childhood fight.
Galactus keeps breaking things in the recording booth.
Mr. Fantastic can't pronounce 'nuclear' right.
Loki is scared that Thor came early for recording since he glued his hammer to the floor that morning.
Ninjas keep attacking Fury.
Jean hates that she always dies.
Mephisto has a lot of friends in Hollywood since he makes deals with people for their astral souls...
Spider-Man can be quite snarky in this game, and it's always hilarious.
Fury: You'll be travelling to Mephisto's Realm. It's a dimension of fire and brimstone.
Spider-Man: Why can we never go to a dimension of lonely super models?
"Am I dead? Why does heaven smell like a wet dog? (notices Wolverine) Oh. Never mind."
The endings of MAU2, playing the Anti-SRA path, there will be a dialogue about Deadpool complaining to one of the developers that he was told that the game was suppose to be "Deadpool and his Inferior Friends". In the Pro-SRA path it'll be Hulk in a talk show.
In MAU2, you get a special team bonus if you make a squad with Spider-Man, Deadpool, Iceman, and the Human Torch. The name of the team? 'Just Shut Up, Already!'
The Gecko Insurance audio ad in MUA2 for a superhero insurance policy. Icing in the cake is this rapid bit of legalese at the end.
Policies do not cover Acts of God or gods such as Thor, Hercules, or Loki.
In the second game, Shocker has captured Ms. Marvel and refuses to believe that she came alone. When she says backup is standing right behind him, Shocker mocks her, then turns around to see your team standing behind him. Cue Oh, Crap! from Shocker as he realises he's in for a beating.
One of Hulk's moves in the second game is a charge move where the poor target Hulk runs into gets knocked to the floor, and is then smashed several times before Hulk gets off them. Level it up enough and few enemies will avoid getting OHKO'd by this attack. Even better, it works on most bosses. For best results, use it on Tinkerer for the hilarious image of a non-combatant frail old man being bowled off his feet and wailed on by Big Green.