The No. 2-seeded Federer took down Isner 7-6 (0), 7-6 (6), 7-5, finally breaking him in the last game.
The 6-foot-10 American couldn't break the 17-time Grand Slam champion -- not even once.
"Obviously, John has one of the best serves in the game," Federer said in his on-court interview. "The pace on his second serve [an average of 115 mph] is unreal. You've just got to hang around and not lose your serve.
"I guess that was a key to the match."
As a result, Federer, a five-time US Open winner, is into the quarterfinals for the 11th time in 12 attempts. On Wednesday, he'll play Richard Gasquet, a four-set winner over Tomas Berdych. Federer has won 14 of 16 matches with the Frenchman.
"He's an unbelievable talent, a great shot-maker," Federer said of Gasquet. "I'm aware of the test ahead."
Federer has won all 12 of the sets he has played so far at the Billie Jean King National Tennis Center.
In the crucible of Monday's two tiebreakers, he was superb. Federer, it turns out, has the best winning percentage in tiebreakers among active players (.651), while Isner is second (.644).
Federer's 7-0 mastery in the first set was the first time in 449 career tiebreakers that Isner was shut out. In the second, Federer trailed 5-3 but won three points in succession. The first came on an unnatural backhand return of a 140 mph serve. At 6-all, Federer hit a service winner off a 128 mph offering. The final stroke was a backhand winner that bordered on the ridiculous.
Federer himself had constructed a streak of 78 straight service holds that ended in a third-round victory over Germany's Philipp Kohlschreiber. Oddly enough, it was Kohlschreiber who had last broken Isner here, in a third-round match in 2013.
The No. 13-seeded Isner was trying to reach the second US Open quarterfinal of his career.
How good is his serve? Down love-40 at 3-4 in the third set, he missed his first serve five straight times and still managed to erase three break points and win all five points.
Ultimately, he saved 23 straight break points in the gargantuan span, but Federer finally solved him.
A couple of terrific shots from the Swiss champion led to an errant backhand volley. Federer has now won five of their six meetings, with Isner's only win coming in a Davis Cup tie in Switzerland on clay in 2012.