Police were called to Revolver Upstairs on Prahran's Chapel St after two men attacked another with a knife and a machete before fleeing about 4am on Sunday.
"A 31-year-old Endeavour Hills man sustained life-threatening injuries and was taken to hospital for treatment," Victoria Police said in a statement.
"The pair of offenders fled the area before police arrived, last seen heading along Macquarie Street."
An Alfred Hospital spokesman told AAP the victim was still in a critical but stable condition.
Revolvers Upstairs has cancelled Sunday night's performance by Bag Raiders. (Joel Carrett/AAP PHOTOS)
The nightclub has been temporarily closed and Sunday night's gig by electronic music duo Bag Raiders cancelled, as management released a statement to social media saying it was "heartbroken" by the incident.
"Our thoughts are with all of those affected by this terrible event," the statement read.
"Revolver's highest priority has always been the safety of our patrons, artists and staff."
Revolver Upstairs, founded in 1997, is something of an institution in Melbourne's dance music scene and hosts DJs and bands six nights a week.
The nightclub is open 24-hours-a-day from Friday afternoon until Monday morning on a typical weekend.
The investigation continues.
It is the second nightclub-related knife attack in a number of weeks.
Six men were arrested after an 18-year-old man was stabbed on August 16 outside the Love Machine nightclub, roughly 500m from Revolver, after a brawl spilled out from the club onto the street.
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