Despite dropping his usually fearsome serve twice in the second set, including while serving for the match at 5-3, Isner closed the match out with a break to win 7-6 (7/4) 6-4 against fellow American Ryan Harrison.
The fifth seed faces another home player in the next round after youngster Jack Sock overcame qualifier Bradley Klahn 7-6 (7/3) 6-3.
Sixth seed Fernando Verdasco claimed his first win since the Australian Open, beating wild card Steve Johnson 7-6 (7/5) 6-2.
Qualifier Robby Ginepri beat 2012 semi-finalist Michael Russell 6-2 6-2 for his first main-draw tour-level win in over a year
Paolo Lorenzi and Flavio Cipolla set up an all-Italian last-16 clash courtesy of wins over qualifiers Gerald Melzer and Facundo Arguello respectively.
Lithuania's Ricardas Berankis beat Jesse Levine of Canada 6-7 (3/7) 7-5 6-4 in almost two-and-a-half hours.