The 17-time Grand Slam champion will play French 12th seed Richard Gasquet -- who eliminated Czech sixth seed Tomas Berdych 2-6, 6-3, 6-4, 6-1 -- on Wednesday for a semi-final berth.
"Of course Roger will be the favorite of the match but I will have nothing to lose," said Gasquet, who is 2-14 lifetime against the Swiss star.
Federer, trying to become at 34 the oldest US Open champion since Ken Rosewall in 1970, stretched his US Open win streak over Americans to 12 since losing to Andre Agassi in 2001.
Isner had held in 110 consecutive US Open service games since broken by Germany's Philipp Kohlschreiber in the third round in 2013, but Federer ended that streak on match point to close out the victory in two hours and 39 minutes.
"John has one of the best serves in the game," Federer said. "You have just got to hang around and win your service games."
Federer, seeking his sixth US Open title, also inflicted the first shutout tie-break loss upon Isner.
"I served very well, picked up his second serves," Federer said. "You've got to get a little lucky."