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The complex ecology of free-living amoebae (FLA) and their role in spreading pathogenic microorganisms through water systems have recently raised considerable interest. In this study, we investigated the presence of FLA and amoebae-resisting bacteria (ARB) at various stages of a drinking water plant fed with river water. We isolated various amoebal species from the river and from several points within the plant, mostly at early steps of water treatment. Echinamoeba- and Hartmannella-related amoebae were mainly recovered in the drinking water plant whereas Acanthamoeba- and Naegleria-related amoebae were recovered from the river water and the sand filtration units. Some FLA isolates were recovered immediately after the ozonation step, thus suggesting resistance of these microorganisms to this disinfection procedure. A bacterial isolate related to Mycobacterium mucogenicum was recovered from an Echinamoeba-related amoeba isolated from ozone-treated water. Various other ARB were recovered using co-culture with axenic Acanthamoeba castellanii, including mycobacteria, legionella, Chlamydia-like organisms and various proteobacteria. Noteworthy, a new Parachlamydia acanthamoebae strain was recovered from river water and from granular activated carbon (GAC) biofilm. As amoebae mainly multiply in sand and GAC filters, optimization of filter backwash procedures probably offers a possibility to better control these protists and the risk associated with their intracellular hosts.