In the first American hardcourt warm-up event ahead of next month's start of the US Open, Matosevic beat Dominican Victor Estrella Burgos 6-0, 6-2 while Istomin ousted US lucky loser Rajeev Ram 6-1, 6-4 on Tuesday.
Next up for Matosevic is American Tim Smyczek, who ripped countryman Ryan Harrison 6-0, 6-2.
A victory in that second-round affair would send the Aussie against either US top-seeded defending champion John Isner, who received a first-round bye, or American wildcard Robby Ginepri, who outlasted Ukraine's Sergiy Stakhovsky 7-6 (7/3), 3-6, 6-0.
Istomin booked a second-round date with Slovakian Lukas Lacko, who downed US wildcard Nathan Pasha 6-2, 7-5.
Ukraine's Illya Marchenko ousted Aussie John-Patrick Smith (AUS) 6-4, 6-4 to book a second-round date with Canadian fourth seed Vasek Pospisil.
Dutchman Thiemo de Bakker downed Canada's Steven Diez 6-1, 6-2 and will face South African second seed Kevin Anderson, last year's Atlanta runner-up, in round two.
Germany's Benjamin Becker defeated Aussie Matthew Ebden 6-4, 6-2 and will next face either US lucky loser Alex Kuznetsov or Taiwan seventh seed Lu Yen-Hsun.