American top seed Isner, a two-time champion at Newport, comfortably overcame countryman and qualifier Austin Krajicek 6-3 6-3 on the grass courts.
Isner served 11 aces on his way to claiming victory in an hour and three minutes at the ATP 250 event.
Awaiting Isner in the last eight is seventh seed Jack Sock, who beat fellow American Rajeev Ram 7-5 6-2.
Sock, fresh from winning the Wimbledon men's doubles crown with Vasek Pospisil, made Ram pay for squandering nine break points.
Croatian Karlovic, the second seed, sent down 18 aces in his 7-5 7-6 (7-3) win over Ukrainian Sergiy Stakhovsky.
He faces Dudi Sela in the quarter-finals after the Israeli thrashed French eighth seed Adrian Mannarino 6-3 6-1 in just 50 minutes.
There was better news for another Frenchman in Nicolas Mahut as the fourth seed stayed on track to defend his title.
Mahut beat Australian qualifier Luke Saville 6-4 6-2.
He will face another Australian in the last eight in Sam Groth, with the big server producing 24 aces in a 7-6 (9-7) 7-6 (7-3) win over Tunisian Malek Jaziri.
Veteran Australian third seed Lleyton Hewitt, runner-up the past two years, advanced thanks to a 6-2 6-2 win over Croatian qualifier Ante Pavic.
Hewitt faces sixth seed Steve Johnson next after the American led Tatsuma Ito 6-3 2-0 before the Japanese right-hander retired with a left knee injury.