On 18 June 1996 Lord Voldemort lured Harry Potter to the Ministry of Magic precipitating the Battle of the Department of Mysteries. During the battle, Dolohov was paired with Jugson as the Death Eaters searched for the five students who had come to the Ministry with Harry Potter. They tracked Harry Potter, Hermione Granger, and Neville Longbottom to a room off the Time Room, hitting the three of them with Impediment Jinxes. When Dolohov began to alert the other Death Eaters as to their location, Hermione used Silencing Charm on him to prevent it. He retaliated with an unnamed curse which, despite being weaker than normal, because it was cast non-verbally, severely injured Hermione. With a kick, Dolohov broke Neville's wand and nose and then cornered Harry, wordlessly threatening him. However, he was momentarily distracted, giving Harry the chance to Body-Bind him.

Before several Order of the Phoenix members arrived, Dolohov recovered, and he was able to defeat Alastor Moody in a duel. He placed Neville Longbottom under Dancing Feet Spell and attacked Harry with the same spell he used on Hermione, but most of the harm was blocked by Harry's Shield Charm. Then he tried used Summoning Charm on the prophecy in Harry's hand, but he was pushed by Sirius Black. He duelled Black where he seemed to get the upper hand but was again incapacitated by Harry with Body-Bind before he could hit Sirius with his signature spell.

Presumably, Dolohov was captured in the aftermath of the battle by Aurors and returned to Azkaban.