Mycobacterium bovis, a pathogen of conservation, livestock, and public health concern, was detected in eight species of wildlife inhabiting protected areas bordering endemic livestock grazing lands. We tested tissues from 179 opportunistically sampled hunter-killed, depredation, road-killed, and live-captured wild animals, representing 30 species, in and adjacent to Ruaha National Park in south-central Tanzania. Tissue culture and PCR were used to detect 12 (8·1%) M. bovis-infected animals and 15 (10·1%) animals infected with non-tuberculosis complex mycobacteria. Kirk's dik-dik, vervet monkey, and yellow baboon were confirmed infected for the first time. The M. bovis spoligotype isolated from infected wildlife was identical to local livestock, providing evidence for livestock–wildlife pathogen transmission. Thus we advocate an ecosystem-based approach for bovine tuberculosis management that improves critical ecological functions in protected areas and grazing lands, reduces focal population density build-up along the edges of protected areas, and minimizes ecological stressors that increase animals’ susceptibility to bovine tuberculosis.