Uncle Ben was the closest thing Peter had to a father. His death changed Spider-Man's life. The tragedy taught him the invaluable lesson that with great power must come great responsibility.
Spider-Man could not save Gwen Stacy as the Green Goblin pushed her off the Washington Bridge. Decades after the story's release, Spider-Man still feels responsible for his first love's death.
Aunt May's death broke Peter Parker. In Marvel comics, the idea of losing Aunt May weighs so heavily on Peter that he requests the demon Mephisto to bring her back to life.
Harry Osborn was Peter Parker's best friend. But he succumbed to the same weakness as his father, becoming Green Goblin. Peter felt responsible for letting Harry slip into the madness.
Ben Reilly, created by villainous Jackal, was Peter Parker's clone. His death at the hands of the Green Goblin was tragic because he understood Peter as no one else did.
Captain George Stacy, Gwen's father, was a close ally of Spider-Man. However, he died in the destruction caused by Doctor Octopus.
Captain Jean DeWolff was an ally of Spider-Man who worked closely with the superhero in his early career. Later, Spider-Man partnered with Daredevil to probe her death.