Isner defeated Ryan Sweeting 6-3, 6-4 without a service break while firing 10 aces.
That set up a meeting between Isner and No. 8 seed Bernard Tomic, who advanced with a 6-1, 7-5 victory over Tim Smyczek.
Another quarterfinal match was created with victories by Marinko Matosevic and the 2010 winner of this event, Ernest Gulbis.
Matosevic surprised sixth-seeded Alex Bogomolov 4-6, 6-1, 6-3 and Gulbis outlasted Steve Darcis 6-7 (6-8), 6-1, 6-3.
The remainder of the second round will be played Thursday, including a match between fourth-seeded Andy Roddick and Denis Istomin. Roddick is trying to win two matches in a tournament for the first time this year.
Celebrity Families of 2014 [PHOTOS]