The Belarusian slumped to a disappointing 4-6, 3-6 defeat to rising Czech star Bartunkova in the third round of the WTA 1000 event.
Bartunkova began the match by breaking Sabalenka's serve. Incredibly, the world No. 34 saved five break points in a crucial sixth game to maintain her advantage at 4-2. Although Bartunkova missed three chances in the next game to go up a double break, the Czech held her next two service games to love to close out the first set.
The upstart was the first to break again in the second set to go up 3-1 and never looked back as an out-of-sorts Sabalenka couldn't earn a single break point in the set.
"I focused on myself, to be calm," said Bartunkova in her on-court interview."Aryna is great. She's a legend. She's a Grand Slam Champion. I'm really happy I won the match. It is the biggest win of my career. It was very difficult conditions for today because half of the court was very cold and very windy. And the other side was very hot and the opposite.
"It was very difficult conditions for both of us. It could be better for sure, especially on my serve. But it was because of the wind and it was cold. So, that's why I think my percentage of my serve was low."
Meanwhile, Sabalenka gave credit where it was due.
"She is very talented, and moves well on court," she said.
"She also serves well so you have to convert your chances against her. She can serve really well and to return from shade or sun is tricky. I wasn't good at this today."
Bartunkova will next face Austria's Sinja Kraus for a spot in the quarterfinals. Earlier, Kraus earned a 6-1, 6-4 win over Dayana Yastremska.
Elsewhere, fourth seed Mira Andreeva eased past Polina Kudermetova 6-4, 6-1 to reach the next round..