Victoria's has hit 11, with another worker and the spouses of two other staff members testing positive for coronavirus.
COVID-19 testing commander Jeroen Weimar said a fourth worker had returned a positive result on Thursday afternoon.
The female assistant manager was tested on Wednesday and had been isolating as a close contact of a fellow Holiday Inn worker.
It follows the male partners of two food and beverage workers testing positive earlier on Thursday after being placed into isolation as primary household close contacts.
"We have, at this point, no significant concern," Mr Weimar told reporters.
"It doesn't extend the scope of our investigation."
The outbreak now encompasses four workers, two staff spouses, two released guests and a family of three who contracted the virus overseas.
Mr Weimar said genomic sequencing confirmed the fifth and six cases linked to the outbreak have the highly infectious UK variant of COVID-19.
"It is our working assumption therefore that all 11 cases associated with the Holiday Inn cluster are all of the UK variant," he said.
"We need to be sure we keep running faster than the virus to ensure we run it to ground."
The outbreak has prompted several states to tighten their borders to travellers from Greater Melbourne.
Victorian testing manager Jeroen Weimar addresses the media during a press conference in Melbourne. Source: AAP
South Australia, while Queensland will bar entry to visitors of the city's exposure sites from 1am on Saturday.
Western Australia also announced its hard border to Victoria would be extended for at least another seven days.
In addition to tightening interstate borders, the outbreak has forced the hotel's closure, a pause on a planned increase to Victoria's weekly international travellers cap and multiple health alerts for potential exposure sites.
Authorities suspect a nebuliser, which vaporises medications into a fine mist, may be to blame for the outbreak.
The medical device was not declared by one of the family members in quarantine, who has an underlying health condition and was taken to intensive care on Tuesday.
COVID-19 Quarantine Victoria Commissioner Emma Cassar said the guest would have been taken to a medi-hotel if they had reported it.
More than 135 hotel staff were stood down and told to get tested and isolate at home for 14 days, while 48 guests were moved to the Pullman Melbourne to quarantine for at least another three days.
Chief Health Officer Brett Sutton said it was possible everyone on that hotel floor had been exposed to the virus through the air.
There have been cases of COVID-19 transmission detected across three Victorian quarantine hotels within a week.
Australian Medical Association president Omar Khorshid said the UK strain had "blown open cracks" in hotel quarantine infection controls.
Victorian minister Jacinta Allan said program infection measures were constantly monitored and reviewed when issues arose.
"We're operating this program under the highest, strictest of standards," she told reporters.
Deputy Opposition Leader David Davis said the state government had not learned from shortcomings in the previous iteration of the hotel quarantine program.
"It's clear there are deficiencies," he said.
But Prime Minister Scott Morrison, in Melbourne's outer southeast on Thursday to spruik the federal government's HomeBuilder plan, is not worried about the outbreak.
"I wouldn't be here if I wasn't confident," he told reporters at Officer.
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