Catholic Church workers unearth the remains of a 19th-century church official, discovering a box-shaped urn chained to his casket. This urn contains relics related to Mater Lachrymarum (Moran Atias), the final member of the ancient trio of malevolent witches known as the Three Mothers. Inside the box lies a magical tunic that, when worn by Mater Lachrymarum, significantly amplifies her dark powers.
The urn is sent to the Museum of Ancient Art in Rome, where Sarah Mandy (Asia Argento), an American art restoration student, is employed. Sarah is in a relationship with curator Michael Pierce (Adam James), a single father who is absent from the museum that evening. With the assistance of assistant curator Giselle (Coralina Cataldi-Tassoni), Sarah opens the box, only to uncover the tunic. While Sarah retrieves some translation tools from her office, Giselle falls prey to the demonic minions of Mater Lachrymarum. Upon returning, Sarah finds Giselle disemboweled and makes a frantic escape from the museum, aided by a mysterious disembodied voice that guides her through magically unlocked doors.
After reporting the harrowing events to the police, Sarah spends the night with Michael and his son. Meanwhile, deep in her lair, Mater Lachrymarum wears the tunic, enhancing her dark influence. The following day, Michael visits the Cardinal who had sent the urn to the museum, only to find that the Cardinal has suffered a grave stroke and is now in a comatose state. Before slipping away, the Cardinal managed to write down the name "Mater Lachrymarum," a message that soon catches the attention of observing witches as Michael departs.
As Rome descends into turmoil, marked by mass suicides, murders, and rampant violence, Michael’s son is abducted by the witch coven. Desperate for help, he reaches out to Sarah. Despite her pleas to involve the police, Michael dismisses her concerns and decides to consult a local priest who specializes in exorcisms. Tragically, en route, Michael is captured and brutally killed by the witches, his son suffering a horrific fate at the hands of the growing coven.
While navigating a train station, Sarah is targeted by a new faction of witches intent on pledging allegiance to Mater Lachrymarum. As Sarah is pursued by both the witches and police detective Enzo Marki (Cristian Solimeno), she manages to evade capture with the newfound ability to render herself invisible, a skill imparted by the disembodied voice.
Sarah encounters Marta (Valeria Cavalli), a benevolent witch and friend of Sarah's late mother Elisa (Daria Nicolodi). Marta eventually reveals that the guiding voice is none other than Elisa's spirit, a powerful white witch who once wounded Mater Suspiriorum, the eldest of the Three Mothers, leading to the tragic accident that claimed Sarah's parents. While Mater Suspiriorum and her sister Mater Tenebrarum are now dead, Mater Lachrymarum strives to ignite a new era of magic, commencing with Rome's downfall.
Returning to her city home, Sarah is horrified to see that Mater Lachrymarum’s forces are lying in wait. Seeking refuge at Marta's house, she discovers the grim truth: Marta and her partner have been brutally murdered. In a desperate flight, she encounters Michael, only to realize with dread that he is a corpse, manipulated by Mater Lachrymarum. In a bid for survival, Sarah incinerates Michael's animated form, aided by her mother's spirit, which banishes him to the underworld.
Driven by urgency, Sarah tracks down De Witt, an alchemist suggested by Marta as a potential ally against Mater Lachrymarum. De Witt provides her with a copy of "The Three Mothers," which offers insight into locating Mater Lachrymarum's lair. Detective Enzo, who has been shadowing Sarah, decides to join her as they venture into the catacombs where the cult operates. However, Enzo is soon separated and subjected to capture and torture alongside De Witt.
Sarah is ultimately apprehended and brought before Mater Lachrymarum, now empowered by the ancient tunic. Rallying her strength, Sarah heals Enzo’s injuries and uses a spear to strip the tunic from Mater Lachrymarum. With a determined throw, she casts the tunic into a raging brazier, instigating the lair's collapse and a fatal pillar crushes Mater Lachrymarum. As the catacombs topple around them, Sarah and Enzo manage to emerge above ground, sharing a chilling yet relieved laugh at their narrow escape.
