Builders are proposing to erect 700 new homes on a greenfield site in Scone that would see the Perthshire village grow by around a third of its current size.
But locals fear expansion will raise air pollution, increase traffic congestion, destroy ancient native woodland and threaten rare wildlife.
Now a new survey has shown overwhelming opposition to the scheme.
Construction firm A&J Stephen unveiled renewed plans at a public meeting and is believed to be preparing a formal planning application.
The move has prompted concerned villagers to set up the Scone Study Group (SSG) to examine the latest proposals.
They have accused Perth and Kinross Council (PKC) of ignoring residents’ concerns over the impact of the scheme and potential risks to public health and the environment.
SSG member Jill Belch, a professor at the University of Dundee’s school of medicine, is a member of the group. She has written to Deputy First Minister John Swinney, MSP for Perthshire North, to highlight their concerns.
She says the scheme could destroy important habitat for protected species such as red squirrels and pine martens, as well as rare birds and insects.
“The wood that they propose to chop down is a ‘link route’ for many wild animals from one forest to another,” she said.
“Wildlife experts have confirmed to us that the proposed open space of new houses will mean the loss of many species.”
The group also believes expansion will worsen problems with heavy traffic and increase levels of toxic fumes, which are a major cause of illness and early death.
The nearby city of Perth was declared an air quality management area in 2006 due to high levels of noxious gases, which frequently breach EU air quality rules.
The results of a poll handed out to 2,500 homes in Scone has shown 98 per cent of respondent are against the development – an increase from the 80 per cent who opposed the original plans.
Fellow member Martin Rhodes added: “From what we can see there is a huge groundswell against the scheme, even more so than in 2007.”
The future of the project could hinge on a decision over the proposed £140 million Cross Tay Link Road, which will connect Scone with the A9 near Luncarty.