Uruguay's Pablo Cuevas and Spain's Marcel Granollers were facing off against Britain's Jonny Marray and Canada's Adil Shamasdin in the men's doubles.
After an almost-four-hour, five-set marathon and down a break at 8-9, Cuevas and Granollers were denied a bathroom break by French umpire Aurelie Tourte.
A witness accused Cuevas of threatening to relieve himself into a ball can under the cover of a towel, prompting a code violation for unsportsmanlike conduct.
The accusation did not go down well, and when a second violation was issued after Cuevas smashed a ball out of the court in anger, chaos ensued.
The code violation saw the 15th seeds docked a point, putting them down triple match point in the final set.
"He got a code violation for unsportsmanlike conduct," Shamasdin said.
"It was during a changeover and I guess it was something to do with a can or something, I really don't know. I think it has something to do even with bathroom breaks, I'm not really sure."
With perceived injustice hanging in the air, Cuevas and Granollers promptly marched to their seats, sat down, and refused to budge.
Marray and Shamasdin, left standing at the other end of the court, simply looked on with bemusement.
The delay lasted just shy of 10 minutes. Forfeiture looked a real certainty, before Cuevas and Granollers eventually returned to play the final two points, losing 6-3, 4-6, 6-4, 3-6, 14-12.
But once handshakes were out of the way, the protest continued.
Granollers shoved an empty ball can towards Tourte, sans the alleged urine, as the umpire was led away from the fuming Spaniard by security.
Granollers vowed to lodge a formal complaint with the referee's office, saying his English was not good enough to articulate exactly what went down on the court.
"She lied, she lied. She thought one thing that was not true," Granollers said of Tourte.
The rules for men's doubles say players are allowed two toilet breaks during a match. Marray — who won the 2012 Wimbledon doubles title — said his opponents should have been allowed to go.
"I think you should be allowed an extra toilet break if it goes to five sets," he said.
"Are you supposed to go before your own service game? I don't know what the actual rule is. I think you should be allowed to go to the toilet. It's deep in the fifth set. I went a couple of times."
Inadvertently, the pair's protest before wrapping up the match almost caused another unintended consequence.
"I actually had to go to the bathroom at that point, because we were waiting," Shamasdin said.