Amoebae serve as environmental hosts to a variety of mycobacteria, including Mycobacterium avium and Mycobacterium marinum. Mycobacterium shottsii and Mycobacterium pseudoshottsii are waterborne species isolated from the spleens and dermal lesions of striped bass (Morone saxatilis) from the Chesapeake Bay. The optimal growth temperature for these fish isolates is 25 °C. In the present study, amoebae were examined as a potential environmental reservoir for these fish pathogens. Several studies demonstrated that M. avium bacilli replicate within the trophozoite stage and reside in large numbers within the cytosol of the cyst of the free-living amoeba Acanthamoeba polyphaga. Results from the present study showed that M. shottsii, M. pseudoshottsii, and M. marinum bacilli were internalized by A. polyphaga trophozoites within 6 h but that intracellular viability decreased by 2 to 3 logs over 10 days. While an average of 25 M. marinum bacilli were identified by electron microscopy in the cytosol of the cyst, <5 M. pseudoshottsii and no M. shottsii bacilli were observed in this location. All Mycobacterium species examined remained viable but did not replicate after encystment and subsequent 48 h incubation in 4% HCl. This concentration of HCl will kill mycobacteria but will not enter amoebal cysts. Bacterial viability studies within stages of the amoeba life cycle indicate fewer M. shottsii and M. pseudoshottsii bacilli within the trophozoite and cyst stages relative to M. marinum.