The supermarket chain has applied to Harrogate Borough Council to build the almost 40,000 square feet store which it hopes will open in 2023.
An application was submitted in December after a public consultation showed support for the plans, with more than 70% of 190 people who took part giving a positive response.
But residents living next to the site on Electric Avenue have now launched a campaign against the proposals which they see as a threat to wildlife at the site which forms part of Oak Beck.
Resident Jennifer Dance said there are also concerns over the consultation process, as well as the impacts on people's daily lives.
She said: "To local residents' horror Tesco quietly held a public consolation and they claimed 70% were in favour of the development.
"However, only two residents on Electric Avenue were approached or consulted.
"They plan to put the delivery road directly behind our property, so everyday we will hear and see delivery vans whizzing up and down the road.
"The proposed site is home to and feeding grounds for lots of wildlife. Many of the animals visit our garden from the site and I have photographic evidence of some.
"I believe sites like this should be nurtured and cherished, not destroyed and replaced with concrete, steel and tarmac in the name of progress, prosperity and profit."
The residents' group met to discuss the plans this week and have launched a petition which has so far attracted more than 330 signatures.
The petition states residents have seen several animals at the site including roe deer, badgers, foxes, newts, frogs, squirrels, hedgehogs, owls, cuckoos and kingfishers.
The petition added: "We do not need another supermarket in this area, in a five-mile radius from this site there are 14 food stores including Aldi, Asda, Sainsbury’s, Morrisons, Co-op, and express shops.
"We do understand the need to support the new homes being built, but this is not the right location."
Tesco was previously granted planning permission to build a supermarket at the site in 2012, but the plans were never brought forward.
It is now proposing a smaller supermarket with a petrol filling station in a development which it said will create 100 new jobs.
The supermarket chain has responded to the concerns raised by residents in a short statement which said it would listen to all feedback.
Tesco also pointed towards an ecological study carried out by consultants which concluded the site is "dominated by habitats not considered to be of ecological importance". The study also said new habitat creation is proposed to offset the loss of any wildlife areas.
A spokesperson for Tesco said: “We will consider all feedback received on our application and continue to work closely with Harrogate Borough Council.
"The application was submitted with an ecological appraisal which specifically considers these matters.”
A decision on the plans from Harrogate Borough Council is expected around April.
By Jacob Webster, Local Democracy Reporter