Mycobacterium bovis (Mbo) and Mycobacterium avium subsp. paratuberculosis (Map) are two bacteria responsible for bovine diseases, tuberculosis and paratuberculosis respectively. Depending on the species, they can be digested, survive or grow within FLA. Studies have shown that Mycobacterium bovis (Mbo) and Mycobacterium avium subsp. paratuberculosis (Map) can grow in FLA in vitro but few studies reported this interaction in an environmental context. Understanding mycobacterial survival and persistence in the environment is important to better understand their life cycle. Our hypothesis is that FLA can be an environmental reservoir and vector of Mbo and Map. To test this hypothesis, we concentrate our work on farm positive for Mbo or Map in France to isolate and characterise environmental FLA associated with Mbo or Map. Water and soil samples from infected farms were collected to isolate FLA. Total DNA from these FLA was used to detect Mbo and Map DNA using a nested qPCR. Subsequently, the environmental FLA were used to characterize their interaction with Map by infection assays. The nested qPCR allowed us to detect some Mbo and Map DNA suggesting a putative association within the environment. Different environmental FLA were isolated and characterised. Three of them were chosen to investigate the permissiveness by infection assays using a Map-GFP strain. Multiplication of infected FLA was explored by videomicroscopy suggesting that Map-GFP could be transmitted vertically during FLA division. Our work led to isolate many environmental FLA from infected farms, to detect the presence of Mbo and Map DNA in environmental FLA, and to show the permissiveness of three different environmental FLA to Map infection. These results highlight the possible role of FLA as reservoirs of Mbo and Map in the environment.