The world number one was forced to pull out of the Madrid Open ahead of her quarter-final match last week, and was unsure whether to head to Rome for the WTA Premier tournament.
However, Williams showed few signs of discomfort as she powered to a 6-2 6-2 victory over Andrea Petkovic of Germany.
The American was quick out of the blocks and claimed the opening four games, and was on course to match that feat in the second set until Petkovic unexpectedly broke her serve in the fourth game.
But Williams' service game was solid for the remainder of the set, and another game on Petkovic's serve made absolutely sure of victory.
There was no such luck for her sister Venus, who slipped out of the tournament in a 6-4 6-2 defeat to Carla Suarez Navarro.
Suarez Navarro leaps straight into the quarter-finals with that victory, after Simona Halep was forced to withdraw from the tournament with an abdominal injury.
Fourth seed Halep - runner-up in Madrid last week - had beaten Madison Keys 5-7 6-0 6-1 in the second round.
Home favourite Sara Errani safely negotiated the challenge of Russia's Ekaterina Makarova, whose brightly coloured floral outfit failed to inspire an equally dazzling performance as she wilted on the Roman clay, losing 6-2 6-3.
Seeded pair Agnieszka Radwanska and Madrid champion Maria Sharapova both progressed in straight sets against Paula Ormaechea and Monica Puig respectively, while 16th seed Sloane Stephens of the United States lost out to compatriot Varvara Lepchenko, who meets top seed Williams next.
Elsewhere, Flavia Pennetta won a closely fought encounter with Belinda Bencic, Ana Ivanovic edged Alize Cornet over two sets, Francesca Schiavone came from a set down to triumph over Garbine Muguruza, and Christina McHale got the better of wildcard Camila Giorgi having lost the opening set 6-1.
Samantha Stosur cruised into the third round against Elena Vesnina, and, in the final match of the day, Jelena Jankovic advanced against Svetlana Kuznetsova, who retired while trailing 6-2 4-0.