Spider-Man's life is often something of a soap opera, and soap operas are nothing without betrayals. The most impactful and devastating betrayals come from those who are closest to the main character, and indeed Peter Parker has suffered his fair share of betrayal over the decades.
Peter is a person more sinned against in his long run rather than a sinner himself, and he's taken more than a few blows to the heart, from Ben to Gwen to losing his marriage, to name just a few examples. He is a man who has lost much and will continue to lose more. But every day, he gets up and answers a cry for help, because the world needs Spider-Man.
Spider-Man Betrays Venom
In Amazing Spider-Man #362, Spidey asks Venom for help in stopping Carnage. Venom agrees, but with one condition: Venom must be allowed to go free afterward. Spider-Man agrees, but after Carnage is captured, he breaks his promise and locks Venom up as well.
Spider-Man is usually a man of his word, but he really had no choice in this situation as Venom being free meant he would continue to try to kill Spider-Man at every available opportunity. Still, the betrayal made Venom hate Spider-Man even more, adding to the danger the next time Venom would break free. Of course, J. Jonah Jameson got in his dig: "Captain America would've kept his word!”
Robbie Robertson Betrays Spider-Man
In Spectacular Spider-Man #155, Joe "Robbie" Robertson is in jail. His son gets in touch with Spider-Man to tell Spidey that Robbie needs to see him urgently that night in the prison. Feeling a sense of obligation over the decent way the Daily Bugle editor treated him over the years, he goes. He finds Robbie there, but he also finds the killer Tombstone waiting.
Spidey thought he could trust Robertson and considered him a friend, but Robbie's handing Spider-Man over to a murderer very nearly got them both killed. This would greatly affect the way Spidey would interact with the editor in the future, not to mention the way Robertson saw himself after the betrayal.
Boomerang Betrays Spider-Man
During the Secret Empire arc, Boongerang had been instrumental in the resistance and was granted a full pardon for his actions. With a clean slate, he could become the hero he looked like he was born to be. He even became Peter Parker's roommate and started earning his trust. It turned out, however, that he had been working for Kingpin the whole time.
Boomerang fooled everybody. Peter thought he had finally made a friend he could trust and helped him get his life together as he went straight. Boomerang showed a lot of promise as a hero and a person, or at least he would have if he'd actually been on the straight and narrow path. Ultimately, Boomerang betrayed not only Spider-Man but also himself and his own promising life.
Harry Osborn Becomes The Green Goblin
The original Green Goblin was Norman Osborn, father of Peter Parker's friend Harry and killer of Peter's girlfriend, Gwen Stacy. Norman, as the Goblin, was killed during a fight with Spider-Man after the death of Gwen, accidentally spearing himself with his own glider. Harry saw it all happen and blamed Spider-Man for the death of his father. He then assumed the Green Goblin identity in Amazing Spider-Man #136 and fought Spider-Man before being defeated.
Between a breakup with MJ, addiction and overdose, imminent bankruptcy, and the death of his father, Harry had a hard go over the years. The domino effect of devastation in his personal life culminated with his assuming the Green Goblin identity and becoming a true villain. Peter knew his friend was in trouble, but he had no idea how much. Even after Harry turned on him, Peter got help for Harry in the form of doctors and psychologists.
Harry Osborne Becomes Kindred
Harry Osborn, a man of many secrets and once Peter Parker's best friend, is Kindred, the powerful and manipulative demonic villain currently making Peter's life hell in Spider-Man comics. Having died, then risen through the ranks of hell, Harry earned from Mephisto the right to return to death, where he works his plans to make Peter and his father Norman's lives miserable.
Harry succeeds beyond his dreams as he captures and kills Spider-Man, then revives him, killing and reviving over and over, taunting him the whole time and blaming him for his own death. Peter always tried to help Harry through his addictions, mental illness issues and other troubles. Peter was always there for him. As Kindred, Harry repays all that kindness with cruel punishment.
Gwen Stacy Cheats On Peter Parker
Peter Parker receives a letter from the long-dead Gwen Stacy, killed by the Green Goblin, confessing to something. Unfortunately, some of the pages of the letter are missing, and the full truth behind Gwen's confession remains a mystery. Later, it is revealed that Gwen was confessing to having had an affair with Harry, bearing two children.
Many fans would like to leave this story out of continuity. This was one that hurt, even if Peter understood some of the reasons for the affair as well as how broken up Gwen was about it. Still, it ripped away emotional scabs barely healed over and subjected Gwen's children to emotional pain and anger.
Iron Man Hunts Peter Parker
Spider-Man took Tony Stark's side during Civil War, going along with the Superhuman Registration Act and generally backing Stark's every play, even unmasking and revealing himself as Peter Parker. Spider-Man then learned of Stark's secret prisons in the Negative Zone and began to reconsider his support of the Act and of Tony Stark.
Instead of addressing Spider-Man's objections, talking them out reasonably or even just letting Spidey go, Stark instead took the very same lawless actions he said he was against. Iron Man had been someone Peter looked up to and trusted, a fellow hero and an Avenger. With this betrayal, he became the biggest threat to Peter's life.