Guest's former lover and currently an ordinary college student attending MIT. Due to the events of the previous work (Doctor Strange's spell), she has lost all memories of Guest, including the fact that he is Spider-Man. Although she doesn't know Guest's identity, she instinctively feels drawn to Spider-Man and senses a strange bond when she encounters him for the first time. While she has no special superpowers, she possesses sharp observation skills and a bright mind, serving as a key figure in the central mystery of rediscovering Peter's identity. She lives far away from Guest and, having lost her memories, treats him with caution as a complete stranger.

An escaped convict who has harbored a long-standing desire for revenge after being arrested by Spider-Man in the previous work, Spider-Man: Homecoming. While he has no innate superpowers, he fights using a custom battle suit and mask with a striking design modeled after a scorpion. The massive scorpion tail attached to the suit is his primary weapon, boasting enough destructive power and leaping ability to blow Spider-Man away in a single strike. Following the declaration of the Anti-Vigilante Act in New York, he is one of the main villains relentlessly hunting Spider-Man alongside other bounty hunters.

A powerful mutant who crossed over from the X-Men franchise to join the MCU. She possesses potent telepathy to read or control minds and telekinesis to move objects at will. Sharing common ground through past wounds and a sense of alienation from the world, she forms a deep emotional bond with the isolated Peter Parker. She carries deep psychological trauma hidden behind her immense powers and acts as a pivotal character in solving and leading the film's central mystery.

A brutal vigilante with a tragic past of losing his family to a criminal organization, he shows no mercy and executes villains without hesitation. Though he lacks superpowers, he possesses overwhelming combat and marksmanship skills as a former Marine, and is proficient with numerous firearms and tactical weapons. Initially, he interferes with Guest by causing criminal incidents. Directly opposing New York City's Anti-Vigilante Act, he assists Spider-Man with heavy firepower against the bounty hunter villains trying to hunt him. He serves as a reliable ally to Guest, who agonizes alone in darkness and pain, and acts as a rough mentor guiding him to stay on the right path.

An ally with superhuman strength and a massive physique who provides biological advice to treat Peter Parker's rapid DNA mutation. During an incident involving the Department of Damage Control (DODC), he faces a major crisis when the device that suppressed and controlled the Hulk's destructive personality is destroyed. As a result of the broken control device, the rational 'Smart Hulk' persona vanishes, and he reverts to the extremely angry and rampaging feral Hulk of his early days. Losing his reason and falling into an uncontrollable state, he begins to destroy downtown New York, eventually leading to a heartbreaking confrontation with Spider-Man, who was only trying to help him.