An ancient urn chained to a coffin is unhearted by chance by some men at work along the road bordering the Viterbo's cemetery. The urn contains an age-old tunic and some objects belonging to Mater Lacrimarum, the Third Mother (Moran Atias). The only survivor of the Three Mothers - three powerful witches who had been shedding blood and terror for aeons - Mater Lacrimarum (the Mother of Tears) has been hiding in Rome for centuries and her awakening triggers a chain of mysterious and terrible events: the Evil is back to cast its dark shadow over the city.

Sarah Mandy (Asia Argento), a young student of restoration, co-worker and love's interest of Michael Pierce (Adam James), the curator of the Museum of Ancient Art of Rome, is involved in the escalating and increasingly frantic episodes of violence. Sarah tries to run away but she cannot: the Third Mother is looking for her and Sarah is not aware of the fact that her mother Elisa Mandy (Daria Nicolodi) was a powerful white witch brutally killed by Mater Suspiriorum, the witch from Fryeburg.

Helped by the spirit of her mother, by an eminent esoterism academic, Guglielmo De Witt (Philippe Leroy), and by the Chief Constable Marchi (Cristian Solimeno), Sarah realizes that she has no way out and that she must face the impending threat.