A team of agents are chasing geneticist Bertrand Zobrist (Ben Foster) through the streets. Zobrist runs up to the top of a tower when he is confronted by Christoph Bouchard (Omar Sy). Bouchard demands that Zobrist tell him the location of something important and potentially dangerous. Zobrist instead drops himself out the window of the tower and hits two roofs on the way down before smashing to the ground.
Professor Robert Langdon (Tom Hanks) is in a hospital bed. He has a head wound and is suffering from disturbing visions of a hellish scene in which people are shown writhing in pain, while others are burnt and some people have their heads turned backwards. Langdon hears voices and sees someone in a hood, along with other people wearing plague masks. When he awakens, he meets Dr. Sienna Brooks (Felicity Jones), who claims to have been a fan of Langdon's work since she was a child. Sienna tells Langdon he is suffering from retrograde amnesia as a result of the wound. Moments later, an assassin named Vayentha (Ana Ularu) comes in disguised as an officer. She shoots one doctor and then tries to break into Langdon's room to kill him. Langdon and Sienna escape the room, but Langdon is losing blood after the IV is pulled off his arm. They make it into a cab as Langdon passes out.
Langdon wakes up in Sienna's home. He asks her for coffee, despite somehow forgetting the word for it. As she goes to get it, Langdon looks on his laptop to check his emails. He finds one from a man named Ignazio Busoni (Gabor Urmai). The message states that Ignazio is safe, as is something that he and Langdon have apparently stolen, as well as the message "Paradise Twenty Five".
Among Langdon's possessions, he and Sienna find a Faraday pointer, which contains the famous "Map of Hell" by Sandro Boticelli, which was inspired by Dante's "Inferno". It contains letters and Zobrist's name. The two watch a video of Zobrist speaking about his viewpoints on humanity and how the world must be cleansed. This leads the two to discover that Zobrist wanted to unleash a virus worldwide to wipe out billions of people prior to his suicide. Langdon speaks to someone on the phone from the U.S. consulate about being picked up from his room, only to later find out Vayentha and other officers are there for him, forcing him and Sienna to run again.
On the streets, Langdon and Sienna try to figure out the meaning of another statement from the Map of Hell, "cerca trova", which is Italian for "seek and find". Associates from the World Health Organization are now out looking for Langdon too, including Elizabeth Sinskey (Sidse Babett Knudsen). She orders all her men to not trust anyone and to find Langdon by any means necessary.
Vayentha contacts her associates to speak to "The Provost". This is Harry Sims (Irrfan Khan), CEO of a firm called The Consortium. One of his men reports to Sims about what Vayentha has told him. Sims also demands to see a video that Zobrist had sent him to be seen after the virus had been released.
Langdon and Sienna follow clues to the Palazzo Vecchio to see the painting, "The Battle of Marciano", which contains similar writings as the one from the Map. He starts to have more hallucinations and feels a pain in his neck as he remembers being injected with something, leading him to think that he is carrying the virus. Sienna assures him that if he was carrying the virus, everyone around him would probably be dead. They meet with a friend of Langdon's, a museum employee named Marta Alvarez (Ida Darvish). They review security footage and discover that Langdon and Ignazio stole Dante's Death Mask from its exhibit. Langdon must once again run as authorities and museum security get on his tail.
Langdon and Sienna run up to the attic over the Apotheosis of Cosimo I. Vayentha encounters the two up there and tries to kill them, but Sienna pushes Vayentha over, causing her to crash through the ceiling and fall to her death. The WHO officials later arrive and see Vayentha's body, and Sinskey appears to recognize her.
Langdon and Sienna go to the Florence Baptistry where they locate the mask, following the Paradise Twenty Five clue. On the back of it is another hidden message. Afterward, the two are met by Bouchard, who claims to have been the one that met Langdon in Cambridge prior to his amnesia, and also states that Sinskey and her associates are trying to spread the virus. Bouchard joins him and Sienna as they proceed, but Langdon eventually comes to realize that Bouchard is lying to them and that Sinskey was who he met.
Sims and Sinskey meet and discuss the nature of the virus, bringing them to work together to stop it from spreading.
After Langdon deduces that the virus is located in the Hagia Sophia, Sienna betrays him and leaves him, revealing that she was Zobrist's lover and is set to carry on his work. Langdon is then knocked out by an assailant.
We see an extended flashback of Sienna and Zobrist as lovers as he was explaining his philosophy and work to her, which she grew to follow and support. Since they used to play treasure hunts, Zobrist left clues for her to find in case something were to happen to him.
Langdon wakes up and finds he was captured by Bouchard. He was conspiring with Sienna as well. Sims shows up and stabs Bouchard in the neck, killing him. Sims then explains to Langdon that his associates had drugged him with benzodiazepine after he had met with Sinskey and created a fake head wound so that he could help solve the Inferno mystery. Everything that happened in the hospital was also a setup.
Langdon and Sims rejoin Sinskey and locate the tomb of Enrico Dandolo, which leads them to figure that the virus will be exposed at the Basilica Cistern in Istanbul.
The heroes arrive at the Cistern to stop the spread. Sienna kills Sims as the others race to locate the virus. It's underwater in a container set to be detonated. Langdon tries to stop Sienna, but she blows herself up underwater to ensure the virus will spread. Langdon fights one of Sienna's conspirators, but he is shot by the police. Sinskey then manages to keep the virus contained.
The WHO take the virus away. Langdon and Sinskey part ways, but she first returns to him a Mickey Mouse watch that he had lost after their initial meeting.
Langdon returns to the museum to ask a security guard to turn the light up on the Dante mask. The guard enters the room and is stunned to discover the mask in its spot. Langdon just walks away smiling.