In recent decades, free-living protozoa (FLP) have gained prominence as the focus of research studies due to their pathogenicity to humans and their close relationship with the survival and growth of pathogenic amoeba-resisting bacteria. In the present work, we studied the presence of FLP in operational man-made water systems, i.e. cooling towers (CT) and hot sanitary water systems (HSWS), related to a high risk of Legionella spp. outbreaks, as well as the effect of the biocides used, i.e. chlorine in CT and high temperature in HSWS, on FLP. In CT samples, high-chlorine concentrations (7.5 ± 1.5 mg chlorine L⁻¹) reduced the presence of FLP by 63.8 % compared to samples with low-chlorine concentrations (0.04 ± 0.08 mg chlorine L⁻¹). Flagellates and amoebae were observed in samples collected with a level of 8 mg chlorine L⁻¹, which would indicate that some FLP, including the free-living amoeba (FLA) Acanthamoeba spp., are resistant to the discontinuous chlorine disinfection method used in the CT studied. Regarding HSWS samples, the amount of FLP detected in high-temperatures samples (53.1 ± 5.7 °C) was 38 % lower than in low-temperature samples (27.8 ± 5.8 °C). The effect of high temperature on FLP was chiefly observed in the results obtained by the culture method, in which there was a clear reduction in the presence of FLP at temperatures higher than 50 °C, but not in those obtained by PCR. The findings presented here show that the presence of FLP in operational man-made water systems should be taken into account in future regulations.