Two male Florida manatees (Trichechus manatus latirostris) died at the marine aquarium in Inagi City, Tokyo, Japan. Acid-fast bacteria were demonstrated in tuberculoid nodules in the lungs from both manatees. Mycobacterium marinum and M. fortuitum were isolated from one manatee; M. marinum and M. kansasii were cultured from the second animal. This report confirms the pathogenicity and potentially fatal outcome of mycobacterial infection in manatees. In addition, the pathologic response to infection with these mycobacteria in manatees is similar to that associated with Mycobacterium spp. in other animals.