Fernando Gonzalez of Chile defeated qualifier Martin Klizan 6-2, 6-4 in his return from an eight-month injury layoff because of hip surgery.
The big-serving Isner had 18 aces but Mello converted the only break point of the first set to take control of the match. The Brazilian then saved four of the five break points he faced in the second before prevailing in the tiebreaker against last year's finalist.
The 30-year-old Gonzalez dominated the first set, although both players struggled to hold serve in the second. Klizan double-faulted on Gonzalez's second match point.
Gonzalez will play Feliciano Lopez of Spain, who upset eighth-seeded Argentine Juan Monaco 6-4, 7-5.
Lopez made the decisive break in the 11th game of the second set on a wind-swept day at the ATP tour event organized by the family of Novak Djokovic.
"To beat Monaco on clay, you have to be confident," Lopez said.
The 108th-ranked Ukrainian, who converted four of five break points, will play Filippo Volandri. He defeated Serbian wild card Dusan Lajovic 6-1, 6-4.
Somdev Devvarman rallied to beat Mikhail Kukushkin 1-6, 6-4, 6-2, and qualifier Adrian Ungur defeated Ruben Ramirez Hidalgo 6-7 (2), 6-3, 6-4.
Ungur will play the second-ranked Djokovic, who is opening his clay court season on Wednesday after winning 24 consecutive matches this year. He won titles at the Australian Open and hard-court tournaments in Dubai, Indian Wells and Miami.
It's his first tournament back since he pulled out of the Monte Carlo Masters with a knee injury on April 8.