Aims To test the hypothesis that Mycobacterium bovis can persist in the environment within protozoa. Methods and Results In this study, we used a novel approach to detect internalized mycobacteria in environmental protozoa from badger latrines. Acid-fast micro-organisms were visualized in isolated amoebae, although we were unable to identify them to species level as no mycobacteria were grown from these samples nor was M.bovis detected by IS6110 PCR. Co-incubation of Acanthamoeba castellanii with virulent M.bovis substantially reduced levels of bacilli, indicating that the amoebae have a negative effect on the persistence of M.bovis. Conclusions The internalization of mycobacteria in protozoa might be a rare event under environmental conditions. The results suggest that amoebae might contribute to the inactivation of M.bovis rather than representing a potential environmental reservoir. Significance and Impact of the Study Protozoa have been suggested to act as an environmental reservoir for M.bovis. The current study suggests that environmental amoebae play at most a minor role as potential reservoirs of M.bovis and that protozoa might inhibit persistence of M.bovis in the environment.