Background Tularemia is a zoonosis, caused by the bacterium F. tularensis, for which the CHU Grenoble Alpes is the national reference center. Two subspecies of F. tularensis are associated with human tularemia: F. tularensis subsp. tularensis (type A), the most virulent, only present in North America; and F. tularensis subsp. holarctica (type B), less virulent, located in all northern hemisphere and Australia. The last subspecies, F. tularensis subsp. mediasiatica has never been associated with human infections. Human contamination mainly occurs through contact with wildlife animals or arthropod bites. Tularemia can also be a water-borne disease. However, F. tularensis mechanisms of survival in aquatic environments are poorly characterized. Other Francisella species, such as F. philomiragia and F. novicida, are aquatic bacteria and can be occasionally responsible for human diseases in case of immunosupression or of particular aquatic exposure. Methods Firstly, using in vitro models, we investigated interactions of F. tularensis subsp. holarctica, F. novicida and F. philomiragia with amoebae of the Acanthamoeba species. Secondly, we conducted an environmental study by collecting surface water samples in a tularemia endemic region in France. We investigated the presence of F. tularensis and other Francisella species in water using a set of three qPCRs. ISFtu2-qPCR targets Francisella sp.; Tul4-qPCR targets F. tularensis (i.e. F. tularensis subsp. tularensis, F. tularensis subsp. holarctica and F. tularensis subsp. mediasiatica) and F. novicida; and Type B-qPCR targets F. tularensis subsp. holarctica, the only tularemia agent in France. Results In amoeba plate screening tests, all the Francisella species tested resisted the attack by amoebae. In infection model, focusing on strictly intra-amoebic growth, none of the Francisella species tested displayed any intra-amoebic multiplication. In co-culture models, focusing both on intra and extra-amoebic bacterial multiplication, the amoebae favoured Francisella sp. survival over 16 days. In a spring water co-culture model, amoebae again enhanced F. novicida survival and preserved bacterial morphology. Two sampling campaigns were conducted in West of France, in July 2019 and January 2020, allowing the collection of 87 water samples. Among 57 samples of the first campaign, 15 (26.3%) were positive for Francisella sp., nine (15.8%) for F. tularensis and/or F. novicida, and four (7.0%) for F. tularensis subsp. holarctica. The percentages were 83.3%, 80.0%, and 13.3% for the 30 samples of the second campaign. Among the 30 sites sampled during the two campaigns, nine were positive for Francisella sp. both times, seven for F. tularensis and/or F. novicida, and one for F. tularensis subsp. holarctica. Conclusions Our in vitro experiments suggest that amoebae could promote Francisella sp. survival in aquatic environments, including the tularemia agent F. tularensis subsp. holarctica. Our environmental study reveals a high prevalence of Francisella sp., including F. tularensis subsp. holarctica, in the studied aquatic environment. Altogether, our results suggest that the aquatic environment can be a natural reservoir of F. tularensis, and thus could participate in the maintenance of this pathogen in tularemia endemic areas.