The 32-year-old Melzer was able to curb Isner's powerful service game, on his way to a 7-6 (9-7) 6-3 win in the Italian capital.
It took the Austrian just 86 minutes to claim victory, with Melzer's success in the tie-break that ended the first set proving crucial at the ATP 1000 event.
Both players made one break each in the opening set, with American Isner adding 10 aces on the clay.
But the world number 64 showed his class in the tie-break, eventually taking it 9-7, and he never looked back.
Melzer claimed the only break of the second set and ended the match with a better record on serve as Isner faded badly.
There was also further progress for 16th seed Tommy Robredo, who beat Argentina's Juan Monaco 7-6 (7-3) 6-4.
Monaco was not at his best on serve, allowing Robredo to make four breaks, and that was enough for the Spaniard to secure a spot in the second round.
Czech veteran Radek Stepanek fought back from losing the opening set to defeat Spaniard Pablo Andujar 5-7 6-3 6-4 in more than two-and-a-half hours.
And German Philipp Kohlschreiber also had to do it the hard way, toppling Poland's Jerzy Janowicz 4-6 7-6 (8-6) 6-2.
Other results saw Kazakh Mikhail Kukushkin beat Portugal's Joao Souza 6-3 6-7 (1-7) 6-3, while Frenchman Jeremy Chardy defeated Robin Haase of the Netherlands 6-4 6-4.