The world number-nine had produced a strong run on clay, having reached the quarter-finals of the Madrid Open, but she proved no match for resurgent Mladenovic, whose results have improved markedly since the appointment of Naomi Osaka's former coach, Sascha Bajin, in April.
On a busy day of action, Frenchwoman Mladenovic beat Belinda Bencic in the round of 32, then backed that up with her win over Barty.
Osaka won twice at the Internazionali d'Italia as Simona Halep played down fears over her fitness following a defeat to Marketa Vondrousova.
Halep had treatment on a leg injury during the third set of a 2-6 7-5 6-3 loss that confirmed Osaka will be the top seed at this year's French Open.
But the Romanian revealed there were no major issues ahead of attempting to defend her title at Roland Garros this month.
"Yesterday, I didn't do much. I had two days off, so it was a little bit tough to get back to this rhythm, very high rhythm," Halep told the WTA Tour.
"Nothing, like, tight or something broken. It's just a little bit of injury, which in a few days is going to be okay, I'm sure."
Vondrousova returned to the court later the same day, beating Daria Kasatkina 7-5 2-6 6-2 to reach the quarter-finals as the tournament caught up after a Wednesday washout.
OSAKA AT THE DOUBLE
There was some nice symmetry about Osaka's results, the Japanese beating Dominika Cibulkova and then Mihaela Buzarnescu 6-3 6-3.
She will next face the in-form Kiki Bertens, who extended her winning streak to eight matches with victories over Amanda Anisimova and Carla Suarez Navarro, though she needed three sets on each occasion.
In the same half of the draw, Vondrousova goes up against Johanna Konta for a place in the last four.
Konta made it through to the quarters of the event for the first time thanks to triumphs over American duo Sloane Stephens - the seventh seed going out in three sets - and Venus Williams, who had progressed to the last 16 after sister Serena pulled out due to a knee injury.
"I'm playing with a little more variety, a little more awareness and trust in my ability to do different things," Konta said. [I'm] trying to use that in the right proportion during matches."
KVITOVA, MUGURUZA RETIRE IN ROME
Petra Kvitova was forced to cut short her clash with Maria Sakkari when trailing 4-0 in the deciding set, the second seed retiring after treatment on a left calf injury during a medical timeout.
Kvitova showed no concerns during her earlier clash with Yulia Putintseva, however, as she breezed to a 6-0 6-1 result in 54 minutes.
Sakkari will go up against Mladenovic.
Karolina Pliskova survived a scare against Sofia Kenin, though, the fourth seed rallying from a set and a break down to eventually oust the American.
Her opponent will be Victoria Azarenka, who was 6-4 3-1 up against Garbine Muguruza when her opponent retired due to a left thigh injury.