The bacterium Francisella philomiragia has been isolated from environmental samples originating from around the globe. F. philomiragia-related strains cause francisellosis of both farmed and wild fish. In addition, occasional human infections caused by F. philomiragia are found in victims of near-drowning and patients with chronic granulomatous disease. We have shown that F. philomiragia forms in vitro biofilms with increased formation at 25 °C over 37 °C conditions. We found that F. philomiragia can form a biofilm in a co-culture with live Acanthamoeba castellanii, an aquatic amoeba. Interestingly, amoeba-conditioned supernatant has an inhibitory effect on production of biofilm by F. philomiragia, whereas Francisella-conditioned supernatant has no effect on growth of amoebae. We have shown that F. philomiragia can infect A. castellanii after only 5 days of co-incubation and that it infects A. castellanii more quickly than the related species F. novicida does. Our studies point to a potentially overlooked interaction between F. philomiragia and Acanthamoeba. This relationship in the marine lifecycle of F. philomiragia may support the persistence of the bacterium in waterways and its ability to infect fish. An understanding of the persistence of this organism in aquatic systems through biofilm formation and its interaction with Acanthamoeba will be important in developing prevention strategies for this pathogen.