Hunter Valley police served the order on Saturday due to "repeated and continued breaches of the Public Health Order by allegedly allowing, and not taking steps to prevent, unvaccinated people being at the premises".

Police attended the venue numerous times last week and issued the licensee, a 57-year-old man, with three Penalty Infringement Notices (PINs), as well as fining three staff members for breaching the public health orders.

The pub operator has been outspoken on social media against vaccination requirements and vowed to continue operating after first being visited by police, saying in a post on Tuesday: "We are still here cracking along FOR ALL now and always".

Singleton mayor Sue Moore said on Sunday the closure was "inevitable" given they weren't following the health regulations, after being warned repeatedly.

"I know there's some people in town supportive of them, but the ones that have approached me have been more the other way, annoyed and disappointed at what they were doing ... encouraging people to town that are not vaccinated."

Upper Hunter MP David Layzell said people could make personal choices but businesses were expected to comply with restrictions during the transition period if they wanted to operate.

"They are welcome to close the doors until we come to a time that communities can interact safely but we can't have a situation where people don't agree with the restrictions, don't agree with the science of the vaccinations, and still want to operate."