The Burngreave neighbourhood policing team secured a closure order for the property on Burngreave Road because of its links to drugs and prostitution.
There have also been reports of anti-social behaviour associated with the property.
A notice has been placed on the door and anyone who enters is now at risk of prosecution.
South Yorkshire Police said: “After weeks of hard work, the Burngreave neighbourhood team closed down a property on Burngreave Road that was being used to facilitate drug use.
“Other incidents involving suspected prostitution and anti-social behaviour had been reported as taking place at the property.
“A full closure order was granted at Sheffield Magistrates court and a short time later the Burngreave team removed all people from the property and assisted in making it secure.
“Any persons who enter the property are now liable to arrest.”