The pair both teams serve in the opening two games, but on the deuce in the third Zverev was given a time offense.
Umpire Nacho Forcadell claimed the German took longer than the allowed 10 seconds with his opening serve.
But Zverev was furious with the call when he felt he was waiting for Tsitsipas to finish using the towel, causing the delay.
"You had 10 seconds and you didn't earn," preached Zverev.
And the world # 4 replied with a very valid point: "I stood there. I waited for him. Honestly!"
But the official did not have any of it.
"I looked at him and he was ready. You still had 10 seconds," he said.
Zverev then proceeded to argue his side of the story and even called match leader, Terry Armstrong, on the court in bizarre scenes.
"If he doesn't get the towel, it's not 10 seconds. If he's not there, I can't earn," Zverev added.
"I'm at baseline ready. Terry, come here."
Armstrong consulted with Forcadell before telling Zverev that he was in the wrong on this occasion.
Fans inside center court booed as Zverev fumed at the referee.
The 22-year-old tried to get Tsitsipas to confirm his story, but their previous bad blood meant there was no chance of happening.
Zverev and Tsitsipas both highlighted their rivalry in building up the quarterfinals.
They have one winner from their only two ATP meetings.
After Tsitsipas won in Canada last year, Zverev said: "I don't think he played it well.
"I think the fight was completely pathetic at all levels. I am very honest with you (the press). Today was an absolutely pathetic fight, I don't even think he played well."