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Sunderland's Pakistani community set up the mosque at St Mark's Road, Millfield, without planning permission and have spent almost four years fighting to keep it open.
But neighbours raised concerns about noise at prayer times and claimed the street was not suitable for the extra traffic generated by worshippers.
Planners from Sunderland Council agreed and the mosque, which also includes a social and education centre, was originally turned down for retrospective planning permission in January 2006.
The worshippers took their case to the national planning inspector, but the appeal was dismissed.
The council began enforcement action against the unauthorised use of the building, a former Asian food store.
Users of the mosque appealed against the enforcement action, but lost in 2008 and the community was told it must move out by March this year.
However, a fresh planning application was submitted in a bid to overcome the problems, which left protesters furious