WARNING: Spoilers are unmarked.
- Uncle Ben's death, definitely. Peter arrives to find his uncle lying on the sidewalk. He calls his name and Ben hears him ("Peter...?"). Peter says "I'm here, Uncle Ben," but Ben is fading quick and can't even tell his nephew is there. With one last, desperate call out to Pete, Ben dies, leaving Peter to sob over his body.
- The Parting Words Regret makes it even worse. Ben tried to impart a lesson, but Peter is too frustrated to listen. The look on Ben's face after Peter says his line is devastating.Ben: I don't mean to preach, and I know I'm not your father—
Peter: Then stop pretending to be!
- It's even sadder when you consider what really happened in Spider-Man 3. Because Peter let the thief get away with the money, he caused Flint Marko to accidentally shoot Ben. Really, that adds more guilt to Peter's conscience, inadvertently making him responsible for his uncle's death.
- The Parting Words Regret makes it even worse. Ben tried to impart a lesson, but Peter is too frustrated to listen. The look on Ben's face after Peter says his line is devastating.
- Hearing Mary Janes father screaming at Mary Jane and her mother is pretty sad. The arguing between MJs parents doesnt help it either.
- Norman arguing with his Goblin personality when he finds out that Peter Parker, Harry's best friend and admirer of Norman's accomplishments, is the secret identity of Spider-Man. Norman really doesn't want to kill him, yet the Goblin constantly taunts him so much with promise of power that he ends up going through with it. It's a heartbreaking scene and underscores just how conflicted Norman is with what he has done throughout the movie.
- Goblin attacking Aunt May at her home while she's praying to Uncle Ben, all in an effort get to Peter/Spider-Man, by attacking "his heart". Soon after, she's rushed to the hospital, hysteric and traumatized from being menaced by the villain. Just as Peter storms, May, not aware of her nephew's presence, cries "Those eyes! Those horrible yellow eyes!". Peter is appropriately horrified at the realization that Goblin knows who he is, and can attack anyone he cares about. Next on Gobby's hit list was Mary Jane.
- The scene on the bridge, with the Goblin holding Mary-Jane and a cable car of children hostage:
- The kids' supervisor is as scared as they are but trying to keep them calm, all the while knowing that they could die at any moment.
- Spider-Man begging the Goblin to not drop anyone, and he's not snarking. It doesn't work. Then the Goblin attacks him while Spider-Man's holding the cable, and Spider-Man is doing all he can to hold on, slipping several times.
- The citizens of New York coming to help Spider-Man by pelting debris at the Goblin. Throughout the whole movie, Peter has been saving their lives and stopping criminals, never expecting thanks, with Goblin gleefully saying that they will turn on him. Then, while he's trying to keep a cable car of children as well as Mary-Jane from falling into the Hudson, people on the bridge throw all that they have.Bystander 2: You mess with Spider-Man, you mess with New York!
Bystander 1: You mess with one of us, you mess with all of us!
- When Norman dies. "Don't tell Harry."
- To clarify, he doesn't want his son to realize that his father had become a maniacal killer with an alternate personality born from unstable augmentation formula. His last moments were spent begging Peter to keep it a secret.
- It's also a bit telling in that Norman knew he meant the world to Harry, despite how aloof he's been throughout the movie, to the point of even seeming preoccupied in Peter's accomplishments over his own son's. Before leaving for his final showdown with Spider-Man, Norman even promises to try and make amends to Harry. Now it'll never happen.
- Norman's funeral, particularly how bad Harry is taking it.
- The ending. Peter finally has what he's always wanted, the chance to be together with Mary Jane... and he has to reject her to keep her safe.