Ahmad Nadir, 28, and Sarah Abu Lebdeh, 27, have pleaded not guilty to being menacing and offensive when they allegedly said they would refuse to treat Israeli patients and threatened violence against them.
The pair are due to face a trial at the end of August, but the rejection of key footage has delivered a blow to the prosecution case.
Judge Michael McHugh on Tuesday ruled videos and any other recordings of the conversation could not be admitted as evidence in the upcoming trial.
"I've come to the firm view that the video evidence must be excluded from each of the trials of the applicants," he told Sydney's Downing Centre District Court.
Lawyers for the pair earlier argued the nurses were recorded without their consent while at Bankstown Hospital in southwest Sydney, amounting to an illegal invasion of their privacy.
One of the reasons given by Judge McHugh for ruling out the video evidence was the already broad publication of the material online and in the media.
"The alleged utterances of the applicants during the chat room interactions are on their face at the very least likely highly disturbing to right-minded people," he said.
Israeli national Max Ilinsky, popularly known as Max Veifer, recorded a video of his conversation with the nurses on the platform ChatRoulette in 2025.
The platform, known as Chatruletka in Israel, randomly matches people from across the world to facilitate conversations.
"This man (Mr Veifer) is running his own private ... vigilante activity," Mr Nadir's barrister Greg James KC previously said.
"He doesn't care what legal regime may prevail to obtain the recordings."
Crown prosecutor Justin Hannebery KC previously argued the online chat did not have the same expectations of privacy because of the random nature of the connection between the nurses and Mr Veifer.
"Not all private conversations are created equal," Mr Hannebery said.
"It isn't exactly (a chat) with a close personal friend where that expectation might be regarded as absolutely heightened."
The crucial nature of the evidence in the prosecution case outweighed any questions of how it was obtained, the prosecutor said.
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