Mark, 53, and Kim Liddiard, 56, who have lived in their home on Strathallan Estate for eight years, said they have not been consulted about the new venue and only found out about the move through media reports.
T in the Park - which attracts 85,000 revellers - is moving to the 1,000-acre estate in Perthshire after safety fears were raised over a pipeline at Balado. But the couple, along with other residents in the 30 homes on the estate, feel the festival's move could have a "significant effect" on wildlife.
The picturesque area is teeming with wild animals -including bats, nesting ospreys and kites - while the Machany Water, which splits the estate in half, is an important site for wild salmon spawning.
Mrs Liddiard said: "It was a real shock as to what was going on. Nobody had a conversation with us.
"There's real concern the Machany Water is going to get severely damaged. The reason some people come to the area is for wild salmon fishing.
"We've got nesting ospreys on the estate - they would also be disturbed by a concert. There are a number of protected animals. We can watch ospreys, kites and skylarks." David Summers from the Tay Salmon Fisheries Board, said: "We would like to know what things are planned around the stream and what precautions would be taken to ensure there is no pollution."
Festival organisers DFC have not been required by Perth and Kinross Council to lodge a planning application for the annual event.
Any landowner can temporarily change the use of their land if it is for less than 28 days in a year without needing planning permission unless it has a "significant effect on the local environment".
A spokeswoman for Scottish Natural Heritage said considerations would have to be taken with nesting ospreys in the areas as it is an offence to disturb them while nesting.