The Swiss second seed, winner of a record 17 grand slam titles, beat 13th seed Isner 7-6(0) 7-6(6) 7-5 to set up a last eight clash against 12th-seeded Frenchman Richard Gasquet, who ousted sixth-seeded Czech Tomas Berdych in four sets.
The world number two will be contesting his 46th grand slam quarter-final.
Federer, who never lost his serve, held his own from the service line to thwart Isner and used his returning skills to derail a pair of long streaks enjoyed by the American.
The 34-year-old Federer closed out the two hour, 39 minute fourth-round match by breaking the six-foot-10-inch (2.08 metres) Isner's serve, ending the American's string of 108 consecutive service holds at the U.S. Open, dating back to the beginning of last year's championship.
His perfect scoreline in the opening set tiebreak snapped an even longer Isner streak.
In 429 tiebreakers played by Isner, he had never dropped one 7-0.
"Seven-love obviously is a pretty good score against John," said Federer.
(Reporting by Larry Fine; Editing by Sudipto Ganguly)
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