Police were called to the Area Sheffield bar on Burgess Street, near Barker's Pool, at 2.20am this morning following reports that two men had stab wounds.
They discovered one victim had injuries to the back of his head, and the second had a puncture wound to his lung.
Police said neither injury was life-threatening and both were taken to Sheffield's Northern General Hospital for treatment and are recovering.
Officers took immediate action to impose a closure notice on the bar.
This latest incident comes less than 24 hours after four men were stabbed during fights in the city centre.
However, police ruled out a link between the events of Saturday and this morning.
Detective Inspector Mark Monterio said: "We do not believe that these two incidents inside the Area Sheffield bar are connected to the stabbing incidents which took place in Sheffield city centre in the early hours of Saturday.
“A closure notice was served on the bar soon after the incidents took place, and the bar will stay closed until tomorrow, when we will seek an extension to the closure notice at the magistrates’ court."
Saturday morning's violence ended in five men sustaining injuries in two separate brawls that police believe are linked incidents.
The first fight was reported to police at about 4am in Cambridge Street and resulted in an Asian man suffering cuts and bruises and what police described as 'minor stab wounds'. He was taken to hospital for treatment, but released and did not want to make a formal complaint.
At around 5.53am there was a second brawl in Carver Street, in which three Polish men were stabbed. All were taken to hospital and one was described by police as being in a 'serious but stable' condition.
Another man was hit over the head with a bottle during the second fight and taken to hospital to be treated for a minor head injury. He was later discharged.
Police arrested two men on suspicion of attempted murder and they are still in custody being questioned this evening.
Two further men arrested for public order offences have been freed from custody. One has been released under investigation while the other faces no further action.
Superintendent Caroline Rollitt said officers are unclear over the motives of those involved and are trawling through CCTV.
Extra police were deployed to the streets on Saturday night as a precaution. Earlier in the day crime scene investigation officers in forensic gear were seen clearing away white items of clothing with what appeared to be blood on them.
The spate of incidents promoted residents and bar workers to express concern about feeling unsafe and 'on edge' in the city centre.
Jessica Walton, aged 24, who lives on West Street, said: "I have only just moved here so it puts me a bit on edge.
"My family members visited and saw all the police there and were shocked."
Helena Trabucchi, aged 20, who works at the Tap and Tankard in Cambridge Street, just yards from one of the incidents, said: "We couldn't get to the pub on Saturday morning because of the police cordon. It's a little scary."
Anyone with information should call police on 101.