The 1 hour, 37 minute match, played in humid conditions with peeks of sunshine, had few extended points.
"When I'm able to inch ahead in tiebreakers it gives a lot of confidence, especially when I know the match is on my serve at that point," Isner said.
Karlovic played his first ATP tournament since being sidelined with spinal meningitis from late April to early May. He called for trainer early in the opening set, moving his arms from side to side, explaining what was wrong while sitting in his chair.
"I was dizzy a little bit and it was difficult to focus," the 34-year old said. "I was a little worried that it was the virus or something. It wasn't. I was tired. I wasn't used to playing that many matches."
Karlovic, ranked 167 coming into the week, was satisfied with how he played in the tournament.
"I was just happy I was here," he said. "If anyone told me I'd be in the quarters, I would say he's nuts."
Isner closed out the first tiebreaker with an ace.