In December, the government announced finalised plans for a cull, initially in pilot areas, as a way to curb the spread of tuberculosis in cattle.
In applying for judicial review, the Badger Trust says culling will not stop TB and may in fact help spread it.
Other campaign groups are considering action under the Bern Convention, which protects European wildlife.
The government's plans are likely to result in farmers funding contractors to shoot badgers in a number of areas of England, with two initial pilots in west Gloucestershire and west Somerset taking place later this year.
"We have identified some serious flaws in the way by which the Secretary of State [Caroline Spelman] reached her decision to cull badgers," said Gwendolen Morgan of Bindmans solicitors, lawyer for the Badger Trust.
"Given that Defra's proposals come at an enormous cost to farmers, and threaten to prompt rather than prevent the spread of disease, we hope that this ill-conceived decision will be struck down by the court."
She pointed to government projections that culling would reduce TB incidence by 12-16% over nine years.
TheProtection of Badgers Actsays licences to kill can be granted for "preventing the spread of disease" - and the trust argues that the slow-down in the rate of increase, or "reduction in new incidence", projected by Defra does not qualify as "prevention".
The government's plan involves having Natural England issue culling licences. The Badger Trust says the guidance given to Natural England is unlawful.
Potentially the most important element of its case concerns the methods used to kill badgers.
In the landmark UK study, the Randomised Badger Culling Trial (RBCT), badgers were trapped before being shot.
However, the government plans to allow licensed contractors to shoot badgers as they roam - "free shooting" - which is likely to be less efficient and to increase disruption of badger families.
The RBCT showed that when badger's social groups are disrupted, they roam further, carrying the TB bacterium to more farms.
For this reason, the trust argues that after the pilots, farmers may be forced to resort to trap-and-shoot, because free shooting will either prove ineffective or a hazard to public safety.
That would escalate the cost by anything up to a factor of 10 - and the Badger Trust says this possibility damages the government's economic case for a cull.
As the majority of new cases arise through infection from other cattle rather than from badgers, the trust says the government should simply invest in stricter cattle control measures.
Regulations on farmers were recently tightened and testing regimes intensified - but the National Farmers' Union, the government and other proponents believe culling is needed in badly affected areas in addition to existing measures.
In 2010, 25,000 cattle were slaughtered as a result of TB infection, and the disease is costing the UK taxpayer in the region of Â£100m per year.
They also argue that culling would in the end benefit badgers, as TB infection is seriously debilitating to them.
"Nobody wants to see badgers culled, but no country in the world where wildlife carries TB has successfully controlled the disease in cattle without tackling its presence in wildlife as well," said a Defra spokeswoman.
"Unless further action is taken now, it will continue to get worse."
Badgers are protected under UK law, and also under the Bern Convention on the Conservation of European Wildlife and Natural Habitats.
Campaigners are also considering using this as another weapon to use in the long-running battle over culling.
Last year, wildlife groups began programmes of vaccination, and some animal groups believe this will in the end remove any need for culling.
The Welsh Assembly Government is due to announce its decision on a proposed pilot cull in West Wales early this year.
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The Badger Trust has launched a new legal challenge to the government's plans to cull badgers in England.