is one of the most important water-related pathogens. Inside the water supply systems and the biofilms, interact with other bacteria and free-living amoeba (FLA). Several amoebas may serve as hosts for bacteria in aquatic systems. This study aimed to investigate the co-occurrence of spp. and FLA in drinking water supply systems. A total of 268 water samples were collected from apartment buildings, hotels, and public buildings. Detection of spp. was performed in accordance with ISO 11731:2017 standard. Three different polymerase chain reaction (PCR) protocols were used to identify FLA. Occurrence of varied from an average of 12.5% in cold water samples with the most frequent occurrence observed in hot water, in areas receiving untreated groundwater, where 54.0% of the samples were positive. The occurrence of FLA was significantly higher. On average, 77.2% of samples contained at least one genus of FLA and, depending on the type of sample, the occurrence of FLA could reach 95%. In the samples collected during the study, was always isolated along with FLA, no samples containing in the absence of FLA were observed. The data obtained in our study can help to focus on the extensive distribution, close interaction, and long-term persistence of and FLA. Lack of risk management plans and control procedures may promote further spread of in water supply systems. In addition, the high incidence of -related FLA suggests that traditional monitoring methods may not be sufficient for control.