Isner, the top American in the draw after Andy Roddick's withdrawal, had 20 aces and faced only one break point -- which he lost.
The 6-foot-9 Isner earned his first win on clay this season. The turning point came when a drop shot attempt from Zeballos hit the net to hand Isner his first break of the match and a 6-5 lead in the third set.
The American managed to shake off some nerves and finish off the Argentinean on his third match point.
Colombian qualifier Santiago Giraldo used a series of winners to upset former champion Juan Carlos Ferrero 6-0, 6-3. The 76th-ranked Giraldo hit 32 winners to Ferrero's nine, and required only 17 minutes to win the opening set.
The 12th-seeded Ferrero won the Rome tournament in 2001 and entered with an 18-3 record on clay this season. But Giraldo came in on a run of his own, having beaten tour veterans Nicolas Lapentti and Jarkko Nieminen in qualifying.
The top eight seeded players have first-round byes in singles.
Later on Monday, Federer opened his clay-court season with a rare doubles victory.
Federer never dropped serve and showed off effective volleying skills in his first match inside the new 10,500-seat stadium at the Foro Italico.
Federer isn't a stranger to doubles. He won gold in doubles at the 2008 Beijing Olympics.
Information from The Associated Press was used in this report.