RSPB Scotland said video footage shot at the Perthshire estate showed an osprey attempting to rebuild a nest.
However, festival organiser DF Concerts said the video did not correspond with reports from their ornithologist monitoring the site.
Police were called to the scene on Monday and are investigating.
Consultations are ongoing over a planning application to host the annual music festival at Strathallan, which has drawn a mixed response from locals.
Ospreys are a protected species and cannot be disturbed while nesting.
DF Concerts had hoped to encourage the birds to move to a specially constructed new nest further away from the festival site, having been unable to gain access to remove the old nest entirely.
However, the group's tactic of flying a flag from a cherry picker near the old nest was criticised by nature groups and local campaigners.
RSPB Scotland called in the police after the video footage emerged on Monday, stating that the nest was now "active", and the cherry picker was later removed.
A spokeswoman for DF Concerts said it had been taken down while they awaited further information.
She said: "We've seen a copy of the video referred to by RSPB and it does not correspond with the ongoing monitoring from our ornithologist onsite.
"We are fully aware of and compliant with the legal protocol and as such, we have asked the police and RSPB to seek further information so that the video's authenticity can be fully examined."
Police and nature groups are examining a video purporting to show an osprey attempting to nest near the proposed T in the Park site at Strathallan Castle.