The men were knifed after violence flared at Crystal on Carver Street at around 2.20am.
Sheffield's District Commander, Chief Superintendent Shaun Morley, said the hunt for those involved was underway.
"It is very disappointing that we have had this incident in Sheffield city centre," he said.
"It will be thoroughly investigated and we will identify the offenders and bring them to justice. That work is ongoing now.
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"This kind of incident cannot be tolerated and we will take positive action in terms of the offenders and licensed premises involved in events like this.
"Pubs and clubs are going to have to improve the way they search those going into their premises. Searching regimes and processes have to be looked at if we are going to negate this issue happening again."
A police search of Crystal is underway this morning and CCTV footage from in and around the club is being examined.
Anyone with information should call South Yorkshire Police on 101.