The former world No. 1 was leading 7-5 5-4 having recovered from a 3-0 deficit in the second set when the Ukrainian called for a medical timeout.
Wozniacki, who won her sole grand slam title in Melbourne two years ago, broke Yastremska in the final game of the match to take the second set — and the match — 7-5.
“Obviously, she tried to break my rhythm,” the Dane, who will retire after the Australian Open, told reporters.
“At 5-4 I don’t think there was anything wrong, she was running just fine. That’s a trick that she’s done before and I knew it was coming.”
During the Brisbane International earlier this month, Dutch player Kiki Bertens was asked about Yastremska taking a timeout during their round of 32 match.
“Well, I played her three times and three times it’s happened, so that’s my answer,” said Bertens.
During the final of last year’s Thailand Open, Yastremska called for a medical timeout while trailing 5-2 in the third set against Australian Ajla Tomljanovic, only to rally and win the final set 7-6.
“That medical was trash,” Tomljanovic’s compatriot Nick Kyrgios tweeted during the match.
However, they also state that a code violation for unsportsmanlike conduct may be issued if deemed that gamesmanship is involved.
CNN has contacted the Australian Open and Yastremska, via the WTA, for comment but has not received a response.
Wozniacki will face world No. 78 Ons Jabeur in the third round as she bids to end her career on a high note.