The ante-mortem diagnostics of tuberculosis in wildlife constitutes a major problem due to the difficulties in collecting samples for examination. Therefore, tuberculosis in free-living animals is usually diagnosed post mortem. Ante-mortem examinations for bovine tuberculosis (BTB) were conducted in three European bison living in an acclimatisation enclosure in Bukowiec in the Bieszczady National Park in Poland. The reason for performing these examinations was the documented contact between the aforementioned animals and tuberculous European bison from a free-living herd present in the area. The three European bison were pharmacologically immobilised. Next, their blood samples were collected for immunological studies (interferon-gamma release assays), complete blood count and blood biochemistry tests. Moreover, bronchoalveolar lavage (BAL) fluid from the lower respiratory tract, two nasal cavity swabs and one throat swab were taken from each animal. Tuberculin testing was also performed. The findings of all these examinations indicated the possible infection of the European bison with the tubercle bacillus. Ultimately, the decision was taken to euthanize the animals.