Environmental amoebae have been shown to be a host to pathogenic mycobacteria. Mycobacterium avium, Mycobacterium marinum, and Mycobacterium peregrinum can all grow inside Acanthamoeba and other environmental amoebae. Once ingested by Acanthamoeba, M. avium upregulates a number of genes, many of them similar to genes upregulated upon phagocytosis of M. avium by macrophages. Mycobacteria ingested by amoebae grow intracellularly, acquiring an invasive phenotype, evident when the bacterium escapes the infected amoeba. Once inside of amoeba, it has been shown that mycobacteria are protected from antibiotics and disinfectants, such as chlorine. This chapter describes methods employed for the study of the interaction of M. avium and Acanthamoeba.