Three Gold Coast Titans players, including Bryce Cartwright and Nathan Peats, have been stood down for refusing to have flu shots.
Queensland Chief Health Officer Jeannette Young made the announcement on Friday afternoon after speaking with the NRL about its revised flu-vaccination approach.
The third Titans player has not been named as yet.
"I've had a discussion with them (NRL) this morning and they've stood down those three players at the moment until we work through what it means," Dr Young said.
"All their other players, staff and officials are all vaccinated and that is an amazing outcome. So I'm sure we're going to be able to sort it all out."
The NRL said on Thursday its players were required to be vaccinated for flu as part of its biosecurity protocols, but could be exempted in exceptional circumstances such as on religious, medical or conscientious grounds.
Dr Young said she was waiting for the NRL to review its vaccination-waiver policy, with the latest development threatening to derail the planned May 28 season restart.
Cartwright had said earlier in the week he would not have a flu shot and the Titans confirmed on Friday he had signed a waiver.
Peats responded on social media saying he had a bad reaction to the vaccination in 2012 when he was playing for South Sydney but he intended on getting the flu shot on Friday afternoon.
"I had the option to say yes or no and I chose no for that reason," Peats tweeted.
"If I knew it would blow up I would have said yes straight away."
Morrison backs state governments
Prime Minister Scott Morrison reiterated his support for a "no jab, no play" policy in the NRL following Friday's National Cabinet meeting in Canberra.
But he said it was not up to the Federal Government to make the final call on whether the NRL could proceed with its revised plan.
"[The] state and territories should determine the health requirements," Mr Morrison said.
"If they insist on it, I think that's entirely reasonable.
Mr Morrison's comments were supported by Australia's Chief Medical Officer, Brendan Murphy.
"I think that's really a matter for the relevant state health authorities," Professor Murphy said.
"I personally have a view that everyone should get a flu shot and it's important. But I think that's a matter for the relevant states."
