Ultimate Spider-Man playing chicken with three tidal waves and winning.
How can this be topped? By having him barely outrun a crashing Helicarrier.
Amazing Spider-Man taking advantage of the fact that Juggernaut can break through almost anything and dropping him through the Oscorp building.
And how just before he was hitting him with everything he had in the first-person segment. Take note: if Spider-Man knows how indestructible you are, he won't be holding back.
Noir Spider-Man in general. There's something satisfying about sneaking up on your enemies and yanking them up into a cocoon of webbing, and watching their buddies freak out as everyone starts "disappearing". It's like seeing Spidey take a page from Batman's book.
The final boss battle. Four Spider-Men teaming up to battle Mysterio, who's practically a God now? And winning? Awesome.
Said final battle also ends with an awesome moment in the form of the four Spider-Men beating the tar out of Mysterio before returning to their own dimensions.
There's an achievement/trophy that is one for both the player and whichever Spidey you get it with. To get it you must beat a boss with no damage. That's one for the player. But think about from Spider-Man's view: This is someone who either has given him trouble in the past or he knows nothing of and he utterly curb stomps them. Bonus points if the villain is using their fragment.