Mycobacterium species evolved from an environmental recent common ancestor by reductive evolution and lateral gene transfer. Strategies selected through evolution and developed by mycobacteria resulted in resistance to predation by environmental unicellular protists, including free-living amoebae. Indeed, mycobacteria are isolated from the same soil and water environments as are amoebae, and experimental models using Acanthamoeba spp. and Dictyostelium discoideum were exploited to analyse the mechanisms for intracellular survival. Most of these mechanisms have been further reproduced in macrophages for mycobacteria regarded as opportunistic and obligate pathogens. Amoebal cysts may protect intracellular mycobacteria against adverse conditions and may act as a vector for mycobacteria. The latter hypothesis warrants further environmental and clinical studies to better assess the role of free-living amoebae in the epidemiology of infections caused by mycobacteria.