In a series of flashbacks played in reverse chronological order it is related that in 1939, Professor Trevor Bruttenholm investigated a series of murders in Eastern Europe. Erzebet Ondrushko, a vampire who bathed in the blood of innocents to stay young, was responsible. She had sold her soul to the Queen of Witches, the goddess Hecate, and had just kidnapped the fiancee of one of the townsmen. When the search party confronted Erzebet in her castle, all members of the party were horribly killed, and Bruttenholm was left to face her alone. He tricked her into the sunlight, effectively destroying her.
In the present day, an elderly Bruttenholm, who is overcome with memories of his encounter with Erzebet, takes a particular interest in a publicity stunt case in the Hamptons on Long Island. A haunting has been reported in a mansion recently purchased by developer Oliver Trumbolt, a friend of a U.S. Senator with hands deep in the BPRD's budget, and considered a low priority. Bruttenhom insists that their most advanced team should go: Liz Sherman, Abe Sapien, Hellboy and junior agent Sydney Leach as well as himself (to everyone's surprise). Bruttenholm does not explain his motives at first.
Ana, a blushing bride-to-be is met on the streets by two elderly women who encourage her to go to her bridal gown fitting, where she meets Erzebet, who had already killed the shop owner and her baby. Ana innocently comments that Erzebet reminds her of her "older" sister, offending her until she notices that Erzabet has no reflection. She screams, and everything goes dark. Later, upon being told that Father Lupescu was the one who convinced Ana to go to her fitting despite the disappearances of the young girls in the village, Erzabet pays him a visit, frightening him with her dark powers and confirming that his faith is not enough to keep her power from affecting the town.
The BPRD team arrives at Trumbolt's site and sets up to investigate the haunting. Despite a few open windows, a creepy lifelike replica of Erzabet and an old, suspiciously familiar-looking groundskeeper, everything seems normal until night falls and they each encounter strange ghostly apparitions, culminating in dozens of spirits of Erzabet's former victims. Excited that he may have found a goldmine, Trumbolt ignores the professor's warnings and is attacked.
Ana goes to visit Father Lupescu who convinces her that she should not fear danger because the church is strong and the faith will keep her from harm. She hurries off to have her fitting done, running into Professor Bruttenholm as she leaves. Bruttenholm attempts to explain to Lupescu about Erzabet's true nature and her allegiance to Hecate, but he becomes offended and turns Bruttenholm away.
Using his abilities to detect metal, Leach finds a secret passageway through the house's cellar, inadvertently coming across Trumbolt's body, drained of blood. The blood has been placed in a bathtub, apparently for Erzabet's revival. Bruttenholm and Liz head for the gardens to stop Hecate and Erzabet's Harpy-Hags from summoning her back from the dead while a werewolf attacks Hellboy and Abe. Abe is knocked unconscious and taken by the harpies for experimentation while Hellboy fights the werewolf, eventually subduing it and revealing that it is Father Lupescu, the groundskeeper whom Bruttenholm had earlier identified.
Meanwhile, Liz and Bruttenholm are attacked, first by wolves, then by Erzabet's withered body, which knocks Liz out and takes Bruttenholm. Hellboy is dropped through a hole in the courtyard and meets Hecate, who is perplexed as to why he helps the mortals, and tries to lure him to the dark side. He bluntly refuses again and again, forcing Hecate to take a physical form to deal with him. After Abe escapes them, the harpies come across Hecate, but one is killed as she violently thrashes with Hellboy and the other escapes. Erzabet bathes in Trumbolt's blood and rejuvenates herself, but she begins to wither and decay again from the Holy Water which Bruttenholm had added to the bath. He breaks off a chair leg and finishes her off. Her death enrages Hecate who brutally attacks Hellboy and badly wounds him, and when he realizes her weakness is the sun, he lures her outside, forcing her back into the darkness of her own realm, defeated.
Bruttenholm is preparing for his trip to Transylvania where rumors have it that there is a vampire on the loose. As he is packing, his colleague comes in and they discuss the "right hand of doom" (Hellboy's stone hand); Bruttenholm asserts that so long as evil exists, good will rise up against it, before revealing an image of Hellboy's right hand, signifying that he will be responsible for the destruction of the world.
Hellboy wakens in his bed, admiring Bruttenholm, his adopted father, before falling back to sleep again.Ron Perlman - Hellboy
Doug Jones - Abe Sapien
Selma Blair - Liz Sherman
John Hurt - Trevor Bruttenholm
Peri Gilpin - Kate Corrigan
J. Grant Albrecht - Oliver Trumbolt
Jim Cummings - Tom Manning
Grey DeLisle - Anna, Harpy-Hag #2
Rob Paulsen - Sydney Leach, Anna's Fiance
Dee Dee Rescher - Harpy-Hag #1
Kath Soucie - Erzebet Ondrushko
Cree Summer - Hecate
James Arnold Taylor - Father Lupescu, Young Trevor Bruttenholm
Ginny McSwain - Voice Director