CLAIMS that badger setts on one of the county's biggest country estates have been filled in could put the controversial cull on hold.
Police officers are investigating a complaint about setts being disturbed at Forthampton, near Tewkesbury.
The joint investigation between Natural England, West Mercia Police and Gloucestershire police started in April.
A spokesman for Gloucestershire Police said: "Evidence of possible offences has been collected, but at this stage work is ongoing to establish if these were carried out by anyone connected with the land or by others without the landowners' knowledge.
"Investigations are continuing and no arrests have been made."
The complaint was filed by Jay Tiernan, from Stop the Cull, who discovered the allegedly filled in setts.
If the allegations prove true, the badger cull in Gloucestershire could potentially collapse.
Forthampton Estate covers about 3,000 acres, around three per cent of the cull zone in Gloucestershire.
If it were to withdraw from the cull, it could take the total land area in the county that has signed up to the cull below the necessary 70 per cent threshold.
The Government has said the cull can begin from June 1, which is aimed at curbing the spread of bovine tuberculosis.
Mr Tiernan put in the complaint last month to the police. He said: "We're focussing on Forthampton.
"It really is our prime target in the Gloucestershire cull zone.
"As it's such a large area and would be the end of the cull if the Forthampton Estate withdrew, it's a priority for us."
Protesters against the cull have been to the footpaths at the estate to survey badger setts as well as looking for any evidence of shooting.
Owners of the estate have refused to comment.
The latest investigations come after Gloucestershire County Council voted to oppose the cull in the county, and will write to Government ministers about preventing the cull on council-owned land.