The men, both in their 20s, were among five injured when violence flared at Crystal on Carver Street at around 2.20am.
The five men were all taken to hospital with knife or slash wounds, with two of the victims said to have suffered life threatening injuries.
They remain in hospital this morning in critical but stable conditions.
The other three men were treated for superficial injuries.
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A police probe into the incident is now underway.
A South Yorkshire Police spokesman said: "A number of people have been injured following an incident in Sheffield city centre overnight.
"At around 2.20am this morning, emergency services were called to Crystal Bar in Carver Street in the city centre, following reports of an altercation in which several people had suffered stab or slash wounds.
"On officer arrival, five men in their 20s were taken to hospital, where three of the men were found to have superficial injuries.
"Two of the men suffered life-threatening injuries and remain in hospital in a critical but stable condition.
"An investigation is in its early stages and officers remain in the Carver Street area carrying out enquiries."
Anyone with information about the incident should call South Yorkshire Police on 101 quoting incident number 317 of January 1.