Free-living amoebae (FLA) are described as environmental reservoirs for some bacteria able to resist their phagocytosis. In the environment, the fate of Mycobacterium bovis (Mbo) and Mycobacterium avium subsp. paratuberculosis (Map) responsible for bovine tuberculosis and paratuberculosis, respectively, remains poorly understood and is considered potentially problematic in the eradication and control of these diseases. We hypothesize that FLA may play a role in the persistence of Mbo and Map in the environment. In this study, 90 samples were collected from herds affected by one or both diseases to investigate the diversity of amoeba and their associated bacteria. Metabarcoding analyses revealed that Acanthamoeba, Copromyxa, Naegleria, and Vermamoeba were the most represented genera of FLA, with Pseudomonadota being the bacteria most commonly found associated with FLA. Although no Mbo and Map DNA were identified by sequencing, traces were detected by ddPCR (digital droplet PCR), specifically targeting these bacteria. In conclusion, we described a wide diversity of FLA and associated bacteria in this environment. It also suggests that Map and Mbo could be associated, even weakly, with FLA in the environment. However, this needs to be confirmed by detecting a highest amount of DNA and, if possible, cultivable Map and/or Mbo associated with these environmental FLA.