- Spider-Girl (Mayday Parker) (First appearance)
- Mary Jane Watson-Parker (First appearance)
- Benjy Parker (First appearance)
- Courtney Duran (First appearance)
- Davida Kirby (First appearance)
- Jimmy Yama (First appearance)
- Moose Mansfield (First appearance)
- Spider-Man (Peter Parker) (First appearance; dies) (Only in flashback)
- Crazy Eight (First appearance)
- The Green Goblin (Norman Osborn) (First appearance; dies) (Only in flashback)
The film acknowledges the events of Spider-Man: The Gathering of Five, while also establishing a divergence in continuity, stating that, during the final battle between Spider-Man and the Green Goblin, the two costumed titans took their fight all the way to Forest Hills, where Spider-Man, after a grueling and bloody melee, was finally able to defeat the Goblin, but seemingly at the cost of his own life, unlike in the original timeline where he survived and continued on as a hero.
Another divergence in the timeline is that, unlike in the original timeline where his daughter, May, died only a few days after being born into the world, this new timeline establishes that she survived, and Mary Jane and Peter even had a son, Benjy, as well, born right around the time of Peter's death.
15 years later, May is beginning her sophomore year of high school. One morning, during her first period class, May begins to feel nauseous and asks her teacher, Mr. Slattery, if she can go to the bathroom, which he grants permission. Upon arriving, May’s head begins to seemingly tingle. The nausea then goes away and May returns to class. During a basketball practice game later that day, during P.E., May, a highly skilled player in the sport, manages to dodge all of her opponents using that tingling sensation that she felt earlier. Once she gets close enough to the basket, she then jumps a whopping 13 feet into the air, throwing the ball into the basket while also shattering the glass backboard. May is then sent to the principal’s office, where he grants her a 3 day-suspension. May goes home in sadness and confusion. Upon arriving, May explains what happened to her mother, and Mary Jane reluctantly tells her to come up to the attic with her. When they get to the attic, Mary Jane opens up a chest at the far side of the room. May is then taken aback with shock and awe when her mother begins showing her father’s Spider-Man costumes. She then explains how her husband, May's father, first became Spider-Man, and recites his line and catchphrase about power and responsibility. Mary Jane then tells May that after she was born, she and Peter hoped that the baby wouldn’t gain spider powers. Upon realizing the gift that has been bestowed upon her, May secretly decides to continue her father’s legacy, and thus, begins to design a costume in secrecy.
Meanwhile, in New York City, an elderly couple is walking down the street when they are approached by a shady man. The person then demands the couple to hand over their personal belongings, to which they refuse. The figure then slits the throat of the man with a type of razor, killing him, causing the woman to then scream in terror before the shaded man stabs her in the chest, killing her instantly. The man then says, “It’s all fun ‘n’ games when you play with Crazy Eight” before fading into the shadows behind him, laughing as he does so.
Days later, in Forest Hills, May finally finishes her costume, modeled after Ben Reilly's costume. Swiping her father’s web-shooters, May goes on patrol all across Harlem, hoping to make a name for herself. May soon encounters a mugger in an alley robbing a woman and easily diffuses the situation. May then spends the rest of the night stopping various street-level crimes in both Queens and Harlem. However, when she returns home, Mary Jane, furious about May risking her life, forbids her from ever being a costumed hero ever again. However, May, feeling her sense of responsibility kicking in, secretly disobeys her mother and continues her acts as a costumed vigilante.
Meanwhile, the serial killer continues his reign of crime and terror, leaving a calling card dubbing himself as “Crazy Eight”. The police desperately attempt to arrest Eight, but fail miserably. Eventually, May learns of Eight’s actions and tries to stop him. May eventually finds Eight, and the two fight. However, May is caught off-guard by Eight’s reflexes and agility and is soundly defeated. Distraught, May goes through the next few days in a depression, feeling herself unworthy of her webs. However, during Slattery's history class, he shows a documentary about the various super-heroes of the past to the students. Seeing her father in action inspires May to try and go back after Crazy Eight.
Meanwhile, Crazy Eight’s rampage continues, eventually reaching a boiling point when the villain massacres a police station. May tracks him to the waterfront and the two have at it. Eventually, May gains the upper hand and, when she views the spirit of her late father, finally defeats Crazy Eight. However, unwilling to let May have the satisfaction of victory, planted bombs in the warehouse beforehand. He then says, “Sayonara, Spider-Girl”, just as the explosives go off. May narrowly escapes and returns home, battered and bruised from the fight, only to find her mother waiting for her once again. However, instead of scolding May for disobeying, Mary Jane instead hugs her with tears and explains that when she saw that her daughter was not in bed when she came to check on her, adding to the fact that the fight was broadcast on the news, she realized that there was no stopping her from the hero life, and thus, reluctantly accepts May’s decision to become a costumed crime-fighter. After May goes to bed, Mary Jane looks at a picture of her husband, and then a picture of a newspaper picture of his costumed alter-ego on the wall, praying for the future to be kind to her daughter, for whatever comes.
One week later, May, now going by the name of “Spider-Girl” after remembering what Crazy Eight had said to her before, stands on a rooftop, watching over New York. May is contemplating whatever her future holds in store. However, these thoughts are put aside when she hears police sirens, prompting her to swing into action.
In a mid-credits scene, it is revealed that Crazy Eight had survived his battle against Spider-Girl, as he is seen walking through a sewer under the city.
In a post-credits scene, at Stark Tower, Tony Stark looks over recent news footage of Spider-Girl’s heroics, and thus, initiates the “Next Avengers Protocols” just as the screen cuts to black.
- The story for the film is primarily inspired by What If... ? #105, the first appearance of Spider-Girl, as well as Spider-Girl #2, the first appearance of Crazy Eight, and Spider-Girl #8.
- Crazy Eight's design was heavily influenced by the design of the Joker from the 2008 film The Dark Knight.