University professor Robert Langdon wakes up in a hospital with a head wound and no memory of the last few days. His last memory is walking on the Harvard campus, but he quickly realizes that he is now in Florence. Sienna Brooks, one of the doctors tending to him, tells him he suffered a concussion from being grazed by a bullet, and had stumbled into the emergency ward. Suddenly, a female contract killer named Vayentha breaks into the hospital, shoots another doctor, and approaches Robert's room. Sienna grabs Robert and they flee to her apartment.
Robert finds a biohazard cylinder in his jacket and decides to call the U.S. consulate. He is told that they are searching for him and want his location. Robert gives them a location near the apartment out of respect for Sienna's privacy. However, when he looks from the apartment window and sees an armed Vayentha pull up to the location he has given. Robert realizes that the U.S. government wants him killed. He opens the cylinder and finds a medieval bone cylinder fitted with a hi-tech projector that projects a modified version of Botticelli's Map of Hell. Suddenly, soldiers raid the building. Sienna and Robert narrowly escape.
Robert and Sienna head toward the Old City, believing the cylinder must have something to do with Dante. However, they find that Florentini police and Carabinieri officers have sealed the bridges and are searching for them. They run into the Boboli Gardens, where a solution to the Map of Hell occurs to Robert: ten letters have been added to the ten layers of the Malebolge, and the layers have been rearranged. Moving them back yields "CERCA TROVA", a reference to The Battle of Marciano by Vasari, located in Palazzo Vecchio. The mysterious soldiers appear again. Robert and Sienna get into the Old City using the Vasari Corridor.
The director of the museum that houses Battle of Marciano, Marta Alvarez, recognizes Robert after he and Ignazio Busoni, the director of the Il Duomo, had met with her the previous night and asked to see Dante's death mask, which sits in a room behind Battle of Marciano. Robert realizes he is retracing his own steps from last night. Marta takes Robert and Sienna to the mask and find that it's gone. They look at security footage and see Robert himself and Ignazio stealing the mask. The museum guards turn on Robert and Sienna. At this moment, Ignazio's secretary calls Robert and tells him that he died of a heart attack, and his last words were "Paradise 25."
Robert and Sienna escape the guards, but the soldiers arrive. They cross the attic over the Apotheosis of Cosimo I, where Sienna pushes Vayentha to her death. Robert connects the phrase "Paradise 25" to the Florence Baptistry, where he and Sienna find the Dante mask along with a riddle from its current owner, a billionaire geneticist named Bertrand Zobrist. A man named Jonathan Ferris, claiming to be from the World Health Organization, comes and helps them escape the soldiers. They follow the riddle to Venice, where Jonathan suddenly falls unconscious. Robert is captured by the black soldiers while Sienna escapes.
Robert is taken to Elizabeth Sinskey, the director-general of the WHO, and receives an extensive explanation of what is going on: Zobrist, who committed suicide the week before, was a mad scientist and Dante fanatic who engineered a new strain of the Black Death to cleanse the world of overpopulation. Elizabeth raided Zobrist's safe deposit box, found the cylinder, and flew Robert to Florence to follow the clues. However, Robert stopped calling in after meeting with Marta and Ignazio, and the WHO feared he betrayed them and was working with Zobrist to unleash the plague. The soldiers were the WHO's SWAT team and never meant to kill Robert.
Zobrist had paid a shadowy consulting group called The Consortium to protect the cylinder until a certain date. When Elizabeth took it away, they were obligated to protect whatever the bone cylinder pointed to. They kidnapped Robert after the meeting with Marta and Ignazio, but Robert hadn't yet solved the whole trial. They gave Robert drugs to erase his short-term memory, created a fake head wound, and staged every event up to this point so that Robert would desperately solve it. Sienna, Vayentha, and Jonathan are all actors working for The Consortium; the call to the U.S. consulate was also staged. The leader of The Consortium, having become aware of the bioterrorism plot, has agreed to cooperate with the WHO.
Sienna goes rogue and The Consortium realizes she was a secret supporter of Zobrist. She learned where the plague is being kept after Robert solved the riddle, and resolves to unleash it manually. Robert, the WHO, and The Consortium team up to stop her by going to the riddle's actual location: Istanbul, where Enrico Dandolo, the Doge of Venice, is buried. At the Hagia Sophia, Robert and the other find the plague is in the Cistern, but discover that Sienna is already there. The bag that holds the plague has been broken, infecting all the tourists inside. Sienna runs out of the Cistern and shouts "fire", which makes the tourists stampede out into the city.
It is discovered Sienna didn't puncture the bag; it was water soluble and had dissolved one week previously in the Cistern waters, meaning that the whole world has already been infected. It is also discovered that Sienna was trying to stop the virus herself, but didn't trust the WHO because samples of that virus would certainly find their way into weapons research. The leader of The Consortium tries to escape WHO custody with help from disguised underlings, but he is caught later by Turkish police. It is implied that The Consortium will be investigated and ruined. Sienna receives amnesty in exchange for working with the WHO to address the crisis, since she is a medical doctor and transhumanist writer.
The virus employs DNA modification to cause infertility in one-in-three humans. The virus has no cure, and even with future technology, changing the human genome back would be hazardous. The human race, therefore, has been forced into a new age of self-understanding.
- Robert Langdon: A professor of symbology at Harvard University and the protagonist of the novel.
- Bertrand Zobrist: The main antagonist of the novel despite not appearing physically because of his suicide prior the events of the novel and appearing only in flashbacks of other characters. A genius scientist and a madman who is obsessed with Dante's Inferno, he is intent on solving the world's overpopulation problem by releasing a virus.
- Felicity Sienna Brooks/FS-2080: A doctor and Zobrist's former lover. She also worked for The Consortium. She helps Langdon find the virus Zobrist created, but her past relationship with Zobrist makes her loyalty to Langdon suspicious until the end of the novel. She was a loyal disciple of Zobrist until she reads his last letter and decided to get his new technology before it can fall into the wrong hands. She believes the World Health Organization will cooperate with other government agencies to use Zobrist's new virus for weapons. She uses The Consortium and Robert to follow the Map of Hell and get to ground zero before everyone else, but she realizes that Zobrist had set off a futile search as he released his virus well beforehand.
- Elizabeth Sinskey: The head of the World Health Organization who hires Langdon to find Zobrist's virus.
- Consortium's provost: The head of The Consortium and the secondary antagonist. He tries to accomplish Zobrist's wishes by securing the location of the virus from Langdon and Sinskey and to divulge a video Zobrist made before his death to the media. When he learns that he was helping Zobrist in a bioterrorist attack, he helps the World Health Organization to find the cure despite being arrested at the end of the novel.
- Vayentha: The Consortium's agent in Florence with orders to follow Langdon. She is killed by Sienna in the Palazzo Vecchio.
- Christoph Brüder: Head of the SRS team (part of the European Centre for Disease Prevention and Control) who is ordered by Sinskey to find Langdon after she lost contact with him.
- Marta Alvarez: An employee in Palazzo Vecchio in Florence who assists Langdon with Dante's death mask. She is pregnant with her first child.
- Ignazio Busoni/il Dumino: The director of Il Duomo in Florence who assists Langdon with Dante's death mask. He succumbs to a heart attack prior to the events of the novel.
- Jonathan Ferris: An agent of The Consortium who pretends to be in league with the World Health Organization. He used fake eyebrows and a mustache to pretend to be Dr. Marconi at the beginning of the novel.
- Ettore Vio: The curator of St. Mark's basilica in Venice.
- Mirsat: A guide of Hagia Sophia in Istanbul.