The two-time champion overcame American countryman Bradley Klahn 7-6 (7-5) 6-2 at the ATP 250 event played on hard courts in North Carolina.
Isner, the top seed and champion in 2011 and 2012, served 13 aces as he continues preparations for the upcoming US Open.
The big-server will face Mikhail Kukushkin after the Kazakh 13th seed battled past Dutchman Robin Haase 5-7 7-6 (7-2) 6-4.
South African second seed Kevin Anderson was a 6-3 2-6 6-4 winner over Frenchman Adrian Mannarino.
While Isner, Anderson and Kukushkin avoided losses, four seeds were sent packing.
Spaniard Tommy Robredo (third seed) was the top-ranked player to exit, beaten 6-1 7-6 (7-0) by Frenchman Nicolas Mahut.
Leonardo Mayer, the Argentine fourth seed, fell to qualifier David Goffin 6-3 6-1 as the in-form Belgian claimed his 24th straight win.
Portugal's Joao Sousa (sixth) suffered a 6-1 3-6 7-6 (7-5) loss to Pole Jerzy Janowicz, who progressed despite 10 double faults.
American Steve Johnson (16th) was no match for countryman Sam Querrey in a 6-4 6-3 loss as the latter delivered 19 aces.
Wildcard Ryan Harrison retired due to injury despite leading Czech seventh seed Lukas Rosol 6-3 1-2.
Italian 14th seed Andreas Seppi fought his way past Argentine Federico Delbonis 7-5 6-7 (4-7) 6-2 and Spanish eighth seed Marcel Granollers crushed Frenchman Paul-Henri Mathieu 6-3 6-3.
Spanish 10th seed Pablo Andujar was untroubled in a 6-4 6-3 win over Dutchman Igor Sijsling and Lu Yen-Hsun, the ninth seed from Chinese Taipei, brushed past Slovenian Blaz Rola 6-4 7-5.
Edouard Roger-Vasselin, the French 12th seed, needed two hours and 47 minutes to beat Kazakh Aleksandr Nedovyesov 7-6 (11-9) 6-7 (5-7) 6-4.
American left-hander and 11th seed Donald Young continued his decent form with a 6-1 6-3 thrashing of Canadian Frank Dancevic.