In the last 7 yr, three different species of terrestrial mammals were diagnosed with Mycobacterium pinnipedii either within one collection or through the introduction of an infected animal from another zoo. The affected species included the Malayan tapir (Tapirus indicus), Bactrian camel (Camelus bactrianus bactrianus), and crested porcupine (Hystrix cristata). In the first zoo, all of these were living in exhibits adjacent to a group of South American sea lions (Otaria flavescens) and were cared for by the same keeper. One infected tapir was transferred to a different zoo and transmitted M. pinnipedii infection to three other Malayan tapirs. The tapirs were tested with various diagnostic methods, including comparative intradermal tuberculin test, PCR and culture of sputum samples, Rapid Test (RT), and multiantigen print immunoassay (MAPIA). The M. pinnipedii infection was confirmed at postmortem examination in all animals. RT and MAPIA showed the diagnostic potential for rapid antemortem detection of this important zoonotic disease.