While the world prevalence of tuberculosis (TB) is increasing in the human population, TB infection remains a real concern in some animal populations all around the globe. Most mycobacteria of the TB complex are able to infect zoo and wildlife species, in which the pathogenesis, receptivity and immune responses vary widely. The diagnostic tools usually applied in domestic animals show limited performance in zoo species, especially when prevalence is low. Conversely, investigations of cell-mediated immunity through in vitro assay of γ-interferon may have numerous advantages, as long as the technical limits are known and can be improved upon. Furthermore, recent tools based on the investigation of humoral immunity seem very promising for the detection of antibodies directed against certain immunogenic mycobacterial antigens in a wide range of species. All these methods are currently evaluated in field studies, despite the difficulties to ensure rigorous validation. The development of these diagnostic tools is also impaired by the prevalence of mycobacteria other than TB also able to infect and create relevant disease in their host. Thus, decisions on positive and suspicious-animals issues should be taken based on the evaluation of the risk of transmission to the rest of the zoological collection, the possible treatment options, animal welfare, conservation considerations and, of course, the zoonotic potential of this pathogen.