The Department for Transport (DfT) announced its preferred corridor for the proposed expressway in September.
The "Route B" corridor starts at the M1-A421 junction in Bedfordshire, runs south of Milton Keynes, across north Buckinghamshire and into Oxfordshire, with options to run east or west of Oxford.
But the Berks, Bucks and Oxon Wildlife Trust says it is unhappy with the government’s failure to conduct a strategic environmental assessment or habitats regulation assessment of the preferred route.
"This means the true environmental impact has not been properly considered, and the public has been denied the opportunity to fully scrutinise the comparative economic, societal and environmental impacts of the options," the campaigners said in a statement.
On 27 September, the group sent a pre-action protocol letter to the Department for Transport seeking a response to its concerns and warning of a potential court challenge.
At the end of last month, the group said it formally initiated formal High Court proceedings by issuing a claim. It added that it expects a judicial review hearing to take place in the new year.
A crowdfunding campaign set up to pay for legal fees has so far raised more than half of its £40,000 target.
The Berks, Bucks and Oxon Wildlife Trust said it has the support of national umbrella group The Wildlife Trusts and organisations including the Royal Society for the Protection of Birds and the Campaign to Protect Rural England.
Tessa Gregory, partner at Leigh Day, who is representing the campaigners, said: "The choice of corridor has wide ramifications not only because of the expressway itself but also because of the planned associated development in the area. The potential effects on wildlife are devastating.
"The public has the right to expect that large infrastructure decisions such as this will be subject to proper environmental scrutiny and full public consultation."
A DfT spokesman said: "The Oxford to Cambridge Expressway will improve transport connectivity and growth across the region, and the benefits will be felt by the whole country.
"Protecting the environment is central to how it is developed – we have been engaging with community groups about developing ideas for a road in this area since 2015, and will continue to do so leading to a consultation on specific route proposals next year."
He added that the government would "undertake a full environmental assessment as expected in law".
In November, a group of 11 MEPs, from the Liberal Democrat, Labour and Green parties, wrote to Grayling to raise similar concerns that the necessary environmental assessments had not been carried out on the proposed expressway route.
Plans for development of the Oxford to Cambridge ‘growth arc’, including a new road link, were proposed in a report from the National Infrastructure Commission published in November 2017. The commission called for one million homes to be built in the region by 2050 "if the arc is to achieve its economic potential".