Mycobacteria from lymph node biopsies of patients with cervical lymphadenitis reporting for tuberculosis treatment in Matany and Moroto Hospitals in the transhumant areas of Karamoja, Uganda were isolated and characterized. The AccuProbe® culture identification kits for Mycobacterium tuberculosis complex (MTC), M. avium complex (MAC) and M. avium were used to identify the isolates. Spoligotyping, IS901 PCR and IS1311 and IS1245 restriction fragment length polymorphism (RFLP) were used to characterize the isolates. Of the 43 biopsies, ten M. avium, seven M. tuberculosis, three M. bovis, and two M. intracellulare were isolated. Two isolates could not be identified with AccuProbe® and from 19 samples no mycobacteria could be isolated. Three isolates with the Beijing spoligotype were identified from the seven M. tuberculosis isolates. The spoligopatterns of the M. bovis isolates had previously been detected in cattle in Uganda. Isolation of members of the MAC group reflects the complex interaction between the transhumant communities, water sources and their cattle. None of the M. avium isolates harboured IS901, and all showed several bands on IS1311 and IS1245 RFLP, in accordance with M. avium subsp. hominissuis. Composite dendrograms of IS1311 and IS1245 RFLP showed that the isolates were similar and identical patterns were found. The isolation of M. bovis confirms the human infection with zoonotic mycobacteria in areas where consumption of raw milk and meat is routine. Isolation of environmental mycobacteria also confirms their increasing role in human disease and the occupational risk of infection in the transhumant ecosystem in the absence of safe drinking water and environmental contamination.