- The death of Harry Osborn.
- "I should never have hurt you. Said those things." "None of that matters Peter. You're my friend....." "Best friend."
- The scene of Sandman getting up in the silo. If the shot of him trying to grasp his daughter's locket doesn't do it, it's the music in the scene.
- Similarly, the scene where he simply... blows away in the wind makes for a rather sad and touching moment, especially his last words signifying that he's finally giving up on saving his daughter and now only wants to spend as much time with her as possibly before the inevitable happens.
- Flint Marko's whole character in this film is sad: His daughter is terminally ill. Flint is desperate and resorts to partnering with a criminal to get money, only to end up arrested for an Accidental Murder - and he was only arrested because he stayed behind to call for help for his accidental victim, while his partner dies. When he escapes from jail, he accidentally falls into a testing site and turned into the Sandman. He then tries to use his powers to just commit one big robbery, just one for the sake of his daughter, but he keeps getting thwarted and left empty-handed by Spider-Man, who almost kills him the second time because he's the nephew of Marko's accidental murder victim. Marko becomes desperate enough to partner with another criminal, a supervillain this time, to kill Spider-Man and stop his interference. Once again his partner dies, and he comes to terms with the fact that everyone now sees him as a monster and that nothing he can do will save his daughter or make up for the bad things he's done. Peter's forgiveness of him is his sole consolation. Damn, is that sad.
- The flashback of Ben Parker's death. Especially when we find out that it was an Accidental Murder. Peter's anger only makes it worse.
Peter: NO! I have no intention of settling down! This man killed my uncle and he's still out there!!!
- Peter going to ask Harry for his help in stopping Sandman and Venom. He is pleading for his friend to help him, only to come face to face with his scarred visage, and his friend replying "Get Out" with half anger, half sadness.
- The scene where Peter hits Mary Jane in his symbiote induced rage and his My God, What Have I Done? reaction.
- Gwen realizing that Peter only went out with her to make Mary Jane jealous, and her tearful apology to Mary Jane.
- As much of a Jerkass as he is, Eddie Brock becomes pretty damn pitiful after losing his job at the Daily Bugle as well as his girlfriend Gwen, leaving him a sobbing wreck who resorts to praying to God to kill Peter Parker.
- Peter and Harry, former friends, at each other's throats. As well as when Harry shields Peter with his body and they make up finally at Harry's death scene.
- Aunt May consoles her nephew after his recent downward spiral due to his usage of the symbiote suit and tells Peter to forgive himself and that she believes that he will set things right again.
- MJ being upset over all the critics of her play lambasting her performance, contextualizing her feelings as "I read these words and it's like my father wrote them." Heartbreaking to see someone who wanted to make a life for herself to escape the words of her Abusive Parent and the negative feelings they gave her, only to feel like she hasn't escaped the abuse at all.
- Peter losing his temper with his landlord who has asked him about his overdue rent. The landlord is clearly crushed, and slowly says that Peter's a good boy, and clearly something is wrong for him to be acting like this.
Peter: "You'll get your rent when you fix this damn DOOR!!"
- It's a bit depressing seeing how head over heels Ursula is for Peter, especially during his symbiote-induced craze. She's barely able to contain herself around him and is lapping up all of his attention, baking him cookies and feeding him. And Peter doesn't actually have feelings for her.
- Penny and the Sand Castle, a deleted scene. Basically, Flint saying goodbye to his daughter before he heads off to engage Spider-Man in what he believes will be a life-or-death battle.
Sandman: I swear to you...you won't stop me again.
- Not a part of the movie itself, but rather the DVD release. The commentary track ends with the cast hoping that they get to make another movie. Ouch.
- Perhaps it's for the best: in a storyboard, the 4th film would have Peter getting evicted from his apartment. Yes, this scene has a sad Ursula in it.
- Four years after the film was released, Cliff Robertson died. It's sad watching his role, which involves flashing back to his character's death, knowing that it was his last.
- Just a year before, Lucy Gordon, the actress who portrayed the newswoman in the climax, committed suicide. Much sadder is that this was pretty much the only film she was recognized for, and her role is not very appreciated.