Opelka, who received a wild card into the tournament, fired 19 aces to improve to 4-0 against France's Mannarino and advance to the last eight in a grass-court tournament for the first time.
Opelka, 26, is back on the tour after prolonged absence in the wake of hip and wrist surgery.
He is playing in his first ATP Tour event since Washington in 2022, when he was ranked a career-high 17th in the world.
Mannarino, the world No. 25 who was the top seed and coming off a first-round bye, fell to 1-5 on grass this year. He had an impressive 12-4 record on the surface in 2023.
Opelka had opened his campaign with a three-set victory over France's Constant Lestienne.
He will next face fellow American Mackenzie McDonald, who beat Britain's Billy Harris 6-3, 6-4.
