A chinkara (Indian gazelle), protected under the act, was allegedly used for a scene in the film at Jam Kunaria village in the border district of Kutch, 375 km from here.
Forest officials were planning to file a case against Aamir, his former wife Reena and his present wife Kiran Rao for violating the law by filming the animal without permission.
Reena was executive producer and Kiran Rao was assistant director for the film that won critical acclaim apart from being a commercial success.
According to the forest officials here, Aamir had sought permission to shoot a scene with the animal, but the request was turned down.
The film, however, shows a chinkara in a scene.
The act prohibits even teasing or chasing a chinkara is punishable under the law.
"We have surveyed the spot on Monday where the film was shot. A detailed inquiry will be conducted and statements of all concerned people will be taken," a forest official said Tuesday.
Meanwhile, a forest beat guard has admitted that he had taken the crew to shoot a chinkara.
After the film's release in 2001, the forest department reportedly carried some preliminary investigation but it was soon given up.
The issue, however, came to light this month when the Gir Youth Nature Club, an environment group, registered a complaint.
The state police was earlier asked to re-investigate poaching charges levelled against another Bollywood star, Salman Khan, who reportedly killed a chinkara while shooting for Sanjay Leela Bhansali's "Hum Dil De Chuke Sanam" in Kutch.