Michael Morbius, a Nobel Prize-winning biochemist, attempts to cure himself of a rare blood disease with an experimental treatment involving vampire bats and electroshock therapy. He instead becomes afflicted with a far worse condition that mimics the powers and bloodthirst of vampirism. Morbius now has to digest blood in order to survive and a strong aversion to light. He gains the ability to fly, as well as superhuman strength and healing abilities.

Morlun is from a race called the Ancients, that feed on "totem" people like Spider-Man. While Spider-Man is learning the true intentions of totemistic hunters from his new friend Ezekiel, Morlun readies for his next hunt: Spider-Man. His original hunt was for Ezekiel who also had Spider powers like Peter.

Jameson is usually the publisher or editor-in-chief of the Daily Bugle, a fictional New York newspaper and now serves as the mayor of New York City. Recognizable by his mustache, flattop haircut and ever-present cigar, he carries out a smear campaign against Spider-Man that has turned much of the city against the hero. He unknowingly employs Spider-Man's alter ego Peter Parker as a photojournalist.

Aleksei Sytsevich is a poor immigrant from Russia who participates in an experiment that bonded a super-strong polymer to his skin and gave him enhanced strength, speed, and stamina.

When Spider-Man rid himself of the alien symbiote costume he acquired during the Secret Wars, it bonds with reporter Eddie Brock, who hates Spider-Man for causing him to lose his job and reputation. The symbiote, known as Venom gives Brock all of Spider-Man's powers, protection from his Spider-Sense and the knowledge of Parker's secret identity. Brock would gain a new symbiote and face-off against Mac Gargan as Anti-Venom.

Originally a normal scientist and ambitious businessman, Norman Osborn uses an experimental formula which gives him superpowers but also drives him insane. When Spider-Man thwarts his plans to take over as crime boss of New York City, he dedicates himself to utterly destroying Spider-Man's life. Their mutual animosity becomes personal when Osborn kills Gwen Stacy, Parker's long-time girlfriend and first true love. He is generally acknowledged to be Spider-Man's arch-nemesis and worst enemy, and also is responsible for the murder of Ben Reilly, the disappearance of Peter Parker's infant daughter, his son Harry's spiral into madness and eventual death, and masterminding the entire Clone Saga.

Carnage was the combination of a symbiote spawned from the original Venom symbiote and the serial killer, and former cell mate of Eddie Brock, Cletus Kasady. He thrived on murder and chaos and was many times stronger than Spider-Man. He was later killed by the Sentry.

Sergei Kravinoff is a maniacal big game hunter known as Kraven the Hunter who seeks to defeat Spider-Man to prove that he is the greatest hunter in the world. Unlike other hunters, he typically disdains the use of guns or bow and arrows, preferring to take down large dangerous animals with his bare hands. He also uses a mystical serum to give him similar strength to Spider-Man, but even without the serum he is a threat to the wall-crawler. Spider-Man proves a frustrating quarry because Kraven continually underestimates the superhero's resourcefulness.

A disgraced stunt man and special effects artist named Quentin Beck dons an extravagant and theatrical costume as Mysterio and seeks to discredit and frame Spider-Man.

While on the run from the law, the escaped convict Flint Marko finds himself on a remote beach during a nuclear weapon testing. His cells are spliced with sand molecules by the radiation, and his body becomes a mass of a sand-like substance.

The elderly Adrian Toomes' turned to a life of crime after his business partner cheated him. With a self-invented anti-gravity pack, wings for faster flying and a birdlike costume, he became the high-flying, lowdown Vulture.

An emotionally stunted man with an inferiority complex, Maxwell Dillon is struck by lightning while working on power lines and discovers that he has a supernatural control over electricity. Donning a green and yellow lightning themed costume, he goes on to battle Spider-Man numerous times.

Dr. Otto Octavius is a respected scientist, one of the world's foremost experts in radiation who used four mechanical arms in his experiments, which bonded with his nervous system after an explosion becoming Doctor Octopus. He combines physical power with mad genius and is a founding member and leader of the Sinister Six.