Muchova eased through the deciding set to earn her third win against a top-10 opponent this year. The 20th-ranked Czech was a semifinalist at the Australian Open, which was won by Osaka in February.
"It was a tough match today, but we had some preparation," Muchova said. "I was ready to play with all I had today and to use all the weapons I could and [do] what I can."
Osaka was coming off a quarterfinal loss to Maria Sakkari in Miami.
"On the third set, I felt I just started off really bad, and I couldn't afford to do that," Osaka said.
Muchova next faces 16th-seeded Sakkari, who defeated Anett Kontaveit 6-3, 6-1.