Third seed Muguruza was triumphant at Roland Garros 12 months ago and the Spaniard, last year's semi-finalist in Rome, proved too good for Julia Goerges in a 7-5 6-4 victory.
Halep, a victor on clay at the Madrid Open on Saturday, was dominant for the most part against Anastasia Pavlyuchenkova but had to regain her composure in a 6-1 4-6 6-0 success.
Just 24 hours on from a gruelling three-set win over Jelena Ostapenko, Muguruza was back in action in the Italian capital.
It looked as though a similarly tricky encounter was in prospect when Goerges was leading 4-1 in the second set.
The French Open champion came roaring back, though, and she is attempting to keep her cool ahead of her defence in Paris.
"I know I'm defending champion," she said. "Hey, not a lot of girls can say that. So I will take it in a positive way and we will see what happens."
Johanna Konta took a proud record into her third-round match with American great Williams, having won their past three encounters.
But on this occasion the fifth seed was second best, as Williams made just 15 errors en route to a 6-1 3-6 6-1 triumph to set up a contest with Muguruza in the last eight.
Simona Halep improved her record against Pavlyuchenkova to 7-0, despite briefly losing momentum in the second set.
More importantly, the Romanian – whose start to 2017 was blighted by illness and injury – has now won eight straight matches as she continues to build towards Roland Garros.
"I feel great," she said. "I feel my game, I feel strong on court, my legs are strong, I'm mentally strong so I feel good on court."
Anett Kontaveit is next up for Halep after the qualifier defeated Mirjana Lucic-Baroni 6-1 6-1.
Elina Svitolina (8), Daria Gavrilova, Kiki Bertens and Karolina Pliskova were also victorious on Thursday.