The film opens with a flashback to 1973 in the Peruvian Amazon. A pregnant researcher named Constance Webb is searching for a rare species of spider with powerful healing properties. She is betrayed by her security detail, Ezekiel Sims, who shoots her and steals the spider for himself. Constance is bitten by a local indigenous spider in an attempt to save her life and the life of her unborn baby, Cassandra, but she dies shortly after giving birth.

The story jumps to 2003, where Cassandra "Cassie" Webb (Dakota Johnson) is working as an emotionally detached paramedic in New York City alongside her partner, Ben Parker (Adam Scott). During a dangerous rescue operation, Cassie is involved in a near-fatal accident, falling into the river and briefly experiencing death. When she is revived, she begins to suffer from mysterious, disorienting visions of the near future, which she initially dismisses as déjà vu.

Cassie soon realizes these are genuine precognitive powers. Her visions lead her to intervene in an attack orchestrated by Ezekiel Sims, who has used the stolen Amazonian spider to gain enhanced physical abilities and limited precognition. Ezekiel is driven by his own recurring visions, which show him being killed in the future by three young women who possess spider-like powers: Julia Cornwall, Anya Corazón, and Mattie Franklin. He intends to murder the teenagers before they can grow into the heroes who will destroy him.

Cassie finds herself the reluctant protector of the three teenage girls, using her burgeoning powers to keep them one step ahead of the relentless Ezekiel. She attempts to hide the girls and later seeks answers about her past and her new abilities, traveling to Peru to find the tribe that helped her mother. There, she learns the truth about Constance's sacrifice and is pushed to accept her destiny as a clairvoyant linked to the "web of life."

In the film's climax, Cassie uses her fully realized clairvoyant powers to guide the girls through a final confrontation with Ezekiel at an abandoned warehouse. She sets up a trap using her knowledge of the future, leading Ezekiel to his death by being crushed by falling debris. During the fight, however, Cassie is severely injured, resulting in her being blinded and paralyzed, fully embracing the classic "Madame Web" physical state from the comics.

The film concludes with Cassie in a wheelchair, now blind but with her powers stronger and clearer than ever. She assures the three girls who have not yet gained their spider powers—that she will mentor and guide them when their time comes. The ending also confirms that Cassie's partner, Ben Parker, is the uncle of a newly born baby named Peter, setting up a clear connection to the wider Spider-Man universe.