In recent years, many nontuberculous mycobacteria (NTM) species have been recognized as pathogens for humans and animals. Molecular diagnostic techniques enable rapid identification and characterization of the different species of mycobacteria. NTM are inhabitants of the soil and water, and enter the body through a defect in the skin or mucosa, causing infections in immunocompromised subjects. Mycobacteriosis has been described in reptiles, especially in chelonians, but the incidence seems to be low in captivity. To date, mycobacteriosis has been described only in three tortoise species. This report involves a captive Hermann's tortoise (Testudo hermanni) diagnosed with cloacal mycobacteriosis and subsequent systemic dissemination by histopathology and PCR. A Mycobacterium sp. belonging to the M. terrae complex was identified as the etiologic agent, and this is the first report of infection in reptiles by this organism.