The Usual Suspects: The line-up scene was meant to be serious but apparently, much to director Bryan Singer's annoyance, the actors kept breaking up (in part because Benicio del Toro kept farting). But ultimately the laughter-filled footage made the final cut.
The Wizard of Oz:Judy Garland had a hard time not laughing at Bert Lahr's histrionics when meeting the Cowardly Lion. After a few takes were ruined, Fleming apparently took her aside, said, "This is serious, darling", slapped her and told her to go do it. She did, but you can see her holding Toto up to hide her face.
Good Will Hunting: Robin Williams' story about his wife's flatulence breaks up both him and Matt Damon, who at one point squeals with delight.
Dungeons and Dragons: Jeremy Irons as the evil Profrion gnaws on the scenery ferociously. When he cries, "My DESTINY!", Bruce Payne, as enforcer Damodar, can't keep a straight face (and it really doesn't seem to be in character).
Dr Strangelove: In a scene where the title character (Peter Sellers) struggles to keep his arm from performing a Nazi salute, Peter Bull, standing beside him as the dour Russian ambassador, can't help smiling. Kubrick must have been enjoying Sellers too much to notice.
Men in Black: While holding and interrogating an alien disguised as a talking dog, Tommy Lee Jones finally breaks into a smile that wasn't hidden by a quick cut.
Shark Attack 3: Megalodon: In his autobiography Anything Goes, John Barrowman wrote that in the scene after his character stalked a shark, he was supposed to say, "I'm wired. Let's go home and make love". The director asked him to improvise something to get a reaction from co-star Jenny McShane so he said, "What do you say I take you home and eat your pussy?" We can see her toothily smile but not break, then there's a quick cut to them in the shower - hey, it worked! Barrowman was surprised the line made the final cut. For the TV version he says he had to redub the line to "What do I say I take you home and watch I Love Lucy?"
Star Wars: Revenge of the Sith: Some have denied it, but it looks an awful lot like Ewan McGregor as Obi-Wan Kenobi can't keep a straight face as he puts his hand over his mouth while saying, "I have seen a security hologram of him killing younglings." George Lucas, never renowned as a director of actors or a writer of dialogue, might not even have noticed.