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Rome is the capital and largest city in Italy. The city has a long history as one of the most influential cities in European history and historically has been a center of political power (first as the capital of the Roman Empire and later as the seat of power of the Roman Catholic Church), and also possesses a rich cultural history.

Rome is listed as a Janus stronghold in The Black Book of Buried Secrets. It also served as a Tomas stronghold in 1960, when the city was selected to host that year's Summer Olympics. It is unknown what the Tomas branch's objective in the city was or if it was related to the Janus presence in the city, or if the Tomas are still active in the city.

Archaeologist Mark Rosenbloom and his children, Jake and Atticus, live in Rome, having moved there after the death of Astrid Rosenbloom, Mark's wife and Atticus's mother.

Amy and Dan Cahill visited Rome while following a hint on the back of Caravaggio's Medusa. The siblings go to the Colosseum (based on a Latin phrase on the back of the painting) and encounter the Rosenbloom children. Amy and Dan discover the original manuscript of Marco Polo's Il Milione and Leonardo da Vinci's Medusa, and escape with the manuscript.

Madrigal agents William McIntyre and Erasmus Yilmaz planned to meet in Rome to discuss the Vespers. However, Vesper One Damien Vesper III murdered McIntyre before Erasmus arrived and stole much of his research regarding the machina fini mundi.

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