Tapirs seem particularly susceptible to mycobacterial infections, especially to tuberculosis caused by M. tuberculosis or M. bovis. In this case series, we report an infection with the non-tuberculous mycobacteria (NTM) species M. avium ssp. hominissuis (MAH) in a group of four (2.2) captive lowland tapirs (Tapirus terrestris). Two female tapirs showed mild respiratory signs such as coughing and mucous sputum production for several years, one juvenile male tapir had to be euthanized due to severe dyspnea, and the adult male only showed mild respiratory signs in 2010. Post-mortem histopathology of the euthanized animal revealed a chronic bronchopneumonia and MAH was detected via culture. Subsequently, the three remaining tapirs were tested further: Serologically the tapirs had high antibody titers against M. avium, but they showed no reaction in the comparative skin test (TST). At several time points the animals were tested for the presence of mycobacteria in different sample matrices including sputum samples, pooled fecal samples as well as swabs from the tapir enclosure to identify potential environmental niches of the pathogen. Moreover, animals were directly sampled using nasal swabs, endoscopic broncho-alveolar (BAL) and gastric lavages. MAH was detected by culture in the sputum samples, in the BAL of the breeding pair, as well as in the swimming pool water and walls, and in swabs taken from the tapir’s sleeping beds. We conclude that the TST is not a useful diagnostic tool to detect MAC infections in tapirs, whereas antibody ELISA and culture from BAL appear more sensitive.This article is protected by copyright. All rights reserved.