Sheffield and Rotherham Wildlife Trust said Smithy Wood, a patch of ancient woodland near junction 35 of the M1, near Chapeltown, would be lost forever if the proposals went ahead.
The group said the area was a designated Local Wildlife Site within the green belt, containing rare, vulnerable species which would not survive elsewhere if uprooted by the development.
“We have been working with local naturalists, South Yorkshire Badger Group and the members of the local community who regularly visit Smithy Wood, to get to grips with exactly what would be lost if the Motorway Service Station proposal went ahead,” said Sheffield and Rotherham Wildlife Trust CEO Liz Ballard.
“We have been collecting records and people have been contacting us from across the city to help – it is clear a wildlife haven is in danger of being lost.”
The South Yorkshire Badger Group found a large forage hole and path on site, both typical of badger activity, as well as a classic badger sett recently used. Bird surveys found nine bird species of conservation concern as well as birds such as great spotted woodpecker, bullfinch, song thrush and kestrel.
Ms Ballard said: “No amount of new tree planting or ‘biodiversity offsetting’ can replace the magical splendour of dappled light on a sea of bluebells in ancient woodland. But trees, bluebells and other flowers, fungi, badgers, other wildlife animals, insects, butterflies and birds which rely on this unique habitat will all be lost too.
“If Smithy Wood is saved from this development it has the potential to be a wonderful haven for wildlife and to continue to provide a place for fresh air and delight for local people. We are urging people to object to this motorway service area because otherwise this patch of ancient woodland will be lost forever.”
She urged people to write to Sheffield Council’s planning department or visit the Trust’s website at www.wildsheffield.com/smithywood.