Objectives: Francisella tularensis is a Gram-negative, facultative intracellular bacterium commonly found in aquatic ecosystems. F. novicida, F. philomiragia, and F. tularensis subsp. holarctica strain LVS can infect a wide range of cells, including cells of Acanthamoeba castellanii. The life cycle of the bacterium and its maintenance in the aquatic microenvironment are poorly understood. The aim of the above research is to contribute to the understanding of the molecular mechanisms of Francisella species adaptation to A. castellanii and the aquatic environment, and to investigate Francisella resistance and survival after entering interactions with amoebae. Material and Methods: The post-entry life cycle resistance of Francisella species in A. castellanii and the effect of cocultivation on the survival of Francisella species in source water were investigated by inoculating bacteria onto culture media and using transmission electron, fluorescence, and light microscopy methods. The mechanisms of cell death of A. castellanii after infection were investigated by flow cytometry and quantitative real-time PCR. Finally, the role of FPO and FNO gene loci was evaluated using the amoeba plate test. Results: The results of this study showed that Francisella species cultured within A. castellanii were more susceptible to the effects of disinfectants compared to bacteria cultured in vitro. In addition, Francisella strains survived longer and in greater numbers in coculture with amoebae than in amoeba-free water. Francisella does not induce apoptosis and necrosis of A. castellanii cells, but the increased expression of Atg16, Atg12, and Atg8 genes during the infection period suggests that the autophagy process plays an important role after amoeba infection. The FPO and FNO gene loci do not play a role during interactions between F. novicida and A. castellanii, suggesting that F. novicida does not rely on the T6SS during interactions with amoebae. Conclusion: Immediately after infection of A. castellanii, Francisella species become more susceptible to the effects of disinfectants in an attempt to resist cell death by amoebae. However, after a prolonged period of cocultivation, bacteria begin to utilise amoebae nutrients and survive better in the presence of amoebae in the long term. A. castellanii plays an important role in the life cycle of Francisella species.