The National Trust for Scotland has submitted a formal objection to a planning application for the course on a protected sand dune system at Coul Links at Embo by Dornoch, Sutherland.
The course has been proposed by US billionaire Mike Keiser whose high-end golf resorts across the world have been frequently down played by his competitor in the field, Donald Trump.
The objection lodged by NTS follows similar protests from organisations such as Scottish Wildlife Trust and RSPB Scotland.
Developers claim that 95 per cent of those who attended recent consultation events - attended by around 200 people - supported the course.
Meanwhile, around 17,500 people have so far signed an online petition against the Coul Links development.
Stuart Brooks, head of natural heritage policy at NTS, said the “beautiful and wild place” should continue to be “protected for the nation”.
He added: “The Coul Links are an example of an increasingly rare coastal habitat of international significance.
“While it is perfectly understandable that local people want and need jobs, we know from the Dornoch Area Community Interest Company that it is the area’s outstanding natural environment that it is the biggest draw for visitors, and this could and should be a positive foundation for sustainable economic development.”
Mr Brooks said the Coul Links and the dune heath sustained a variety of “internationally important wildlife” including plants, birds and insects.
He added: “They represent an increasingly rare part of Scotland’s natural heritage and it is our obligation as a nation to cherish places like this for the long-term in the face of calls for what might well be illusory short-term benefit.”
Developers have claimed that, within 10 years, the course could draw a further 20,000 golfers a years to the Dornoch Firth area which already boasts the Royal Dornoch, Struie, Brora, Golspie and Tain courses.
A report has suggested the course at Coul Links could bring £4.3m a year to the local economy - and £6.7m to the wider Highland area - and support 120 jobs during the first year.
The golf course would cover part of the Loch Fleet Site of Special Scientific Interest (SSSI) which currently extends to 1,232 hectares.
Developers said the golf course will cover 22.7 hectares, with around two thirds of it to fall within the SSSI boundary
A spokesman for STRI Group, the project managers for the Coul Links course, said: “The developers wish to reiterate the nature and extent of the independent survey work undertaken as part of the Environmental Impact Assessment process for the Coul Links project.
“The work has been carried out by independent consultancies, therefore all interested parties can be assured of its accuracy.
“The wildlife and conservation value of the site has been a critical factor for the development team.
“Starting at the inception of the process, specialists were engaged to develop a golf course that minimises initial impact and that will provide long-term biodiversity gain for this special site.
“The support for the project locally has been amazing.
“Alongside our commitment to maintain, protect, and enhance this special site, we are in no doubt that the project will bring both short and long-term benefits to the site and the local community.”
Mr Keiser is working with fellow businessman Todd Warnock, proprietor of the Links House at Royal Dornoch, on the development along with landowner Edward Abel-Smith.
While Mr Trump was developing his course at Menie in Aberdeenshire, he said of Mr Keiser’s prized Bandon Dunes in Oregon: “The views there are no better than what we have, and the dunes themselves are like little toys compared to our dunes.”
Mr Keiser later said the two men were not interested in building the same type of site, and that you wouldn’t find a chandelier in one of his clubhouses.
The planning application now sits with Highland Council with consultation responses due by December 1.