Approximately one in five isolates of Acanthamoeba spp. recovered from clinical and environmental sources are found to harbor obligate, uncultured bacterial endosymbionts of unknown clinical significance. To investigate their possible role in amoebic pathogenesis, four uninfected amoebic strains were exposed to four different endosymbionts, from which 12 stably-infected host-symbiont pairs resulted. Standardized inocula of amoebae with and without endosymbionts were placed on fibroblast monolayers to examine for cytopathic effects (CPEs). Eight to 10 days were required for monolayer effacement by endosymbiont-free amoebae; 5–8 days for amoebae containing Gram-negative rod endosymbionts; and 3 days for two amoebic isolates infected with a Chlamydia-like endosymbiont. All endosymbiont-infected amoebae produced a statistically significant enhancement in CPEs in comparison to uninfected amoebae; endosymbionts alone on monolayers produced no CPEs. This report provides evidence that obligate bacterial endosymbionts are able to enhance amoebic pathogenic potential in vitro by some as-yet unknown mechanism.