Promoter Frank Warren has confirmed there is "no way" Joe Joyce and Daniel Dubois can fight in October if a live audience is not permitted.
Warren had been hopeful boxing fans would be allowed to attend the bout on October 24 with a number of sporting test events taking place throughout England since July.
The UK Government have now suspended any further test events due to a rise in coronavirus cases, pending a review, with increased restrictions on social gatherings coming into force on Monday.
Warren has consequently admitted the highly-anticipated fight will likely be knocked back for a third time, though stressing he's still hopeful the it can take place before Christmas.
"At the moment, we are not really sure what's happening," Warren told IFL TV. "We're waiting to find out whether we'll be allowed people into the venue.
"I think it's looking highly unlikely we'll have any live audience in October."
Asked how the increased restrictions will impact the event, Warren added: "Legally, it's going to make a difference. The venues are going to have to comply with the regulations and rules to put on live events.
"Hopefully we'll get something on before Christmas, but there's no way at the moment we can be running that show in October if we haven't got some live audience."
The showdown between Dubois and Joyce, set to take place at the O2 Arena, was initially scheduled for April 11, before being pushed back to July 11 and yet further to October 24.
With British boxing making it's return in July, spearheaded by both Warren and rival promoter Eddie Hearn, it had appeared as if Dubois and Joyce were finally set to meet.
With the bout looming, Dubois warmed up for the showdown with a comfortable knockout victory over Ricardo Snijders at the end of August.
After a 12-month absence from the ring, Joyce also took a tune-up fight late in July, easing past Michael Wallisch inside three rounds.
With increased restrictions raising further complications once more, however, it appears as if the bout may be delayed for a third time.
