The Badger Trust has lost its Court of Appeal challenge to official culls which could see thousands of the animals lawfully killed.
The ruling by the court means government proposals for a cull in the West of England could go head within days.
It will see farmers and landowners issued with licences to shoot badgers at night with the aim of reducing the badger population by 70% and then to maintain it at 30% for the four-year duration of the scheme.
The Department for Environment, Food and Rural Affairs (Defra) hopes the pilot culls in West Gloucestershire and West Somerset will help tackle bovine tuberculosis (TB) in cattle.
The Trust, which lost a bid at the High Court in July to block the action, is opposed to the move.
It said there was no scientific consensus to prove it would help reduce the incidence of the disease and claimed the scheme could lead to 40,000 animals being "pointlessly killed" during the next four years.
David Wolfe QC, representing the Trust, claimed the two culls would involve killing an estimated 3,400 badgers in each area - each approximately the size of the Isle of Wight - and the long-term intention was to issue licences for up to 10 culls each year.
But Lord Justice Laws, Lord Justice Rimer and Lord Justice Sullivan unanimously rejected the appeal.
After the judgement, Patricia Hayden, vice-chairman of the Badger Trust, vowed to continue the fight against a cull.
She said: "We are very disappointed. We don't know what the next step is, but we will not give up."
Her husband Jeff Hayden, director of finance at the Trust, said police may have to deal with protests.
"There may be protests. We have heard rumours. But the Badger Trust would not condone any illegal activity," he said.
He urged ministers to explore the possibilities of vaccinating cattle and said Trust officials were hoping to hold talks with new Environment Secretary Owen Paterson.
Queen guitarist Brian May, who led a new protest against the Government's plans at a Stop The Cull campaign rally in Bristol, also said the fight against culling badgers would continue despite the defeat in the courts.
He said: "You could kill all the badgers in Britain and it would not stop the problem of bovine TB in cows. It is called bovine TB and it is endemic in cows. You can take away the badgers and you will still have your problem.
"Everybody is of the same opinion - this cannot be allowed to happen. We cannot sacrifice our wildlife in a vain attempt to solve a problem that should be solved by vaccination."
The rally was organised by a coalition of animal welfare charities including the RSPCA, League Against Cruel Sports, Save Me and Humane Society International (UK).
The high profile animal welfare campaigner has called for the vaccination as a humane alternative.
Vaccination programmes are also being trialled in Devon by the National Trust, in Gloucestershire by the Wildlife Trust and in Somerset by the Somerset Badger Group.
TB is the biggest challenge currently facing the cattle farming industry, according to Defra.
In 2011, 26,000 cattle were prematurely slaughtered as a result of the highly infectious disease. The total cost of the cattle losses was estimated at £91m.
It costs the taxpayers £100m a year in testing, inspecting and compensating farmers.
The High Court ruling by Mr Justice Ouseley in the summer was welcomed by the National Farmers Union, the British Veterinary Association and British Cattle Veterinary Association.
