This work studies the characterization of pathogenic free-living amoebae (FLA) from sewage effluents. Some of them, such as Acanthamoeba, Naegleria, Hartmannella, Sappinia, Balamuthia and Paravahlkampfia have been reported as a cause of diseases in humans. Therefore, the study of their habitats and their pathogenicity has become necessary. The population of potentially pathogenic FLA was analysed in five Spanish wastewater treatment plants. Five of the seven FLA isolated were identified as genus Acanthamoeba genotypes T3, T4, T7 and T9. Hartmannella and Naegleria were also isolated. Acanthamoeba demonstrated great thermotolerance and osmotolerance. It was also observed that treatment with sodium hypochlorite showed no significative reduction in the number of amoeba at concentrations of 0–100 ppm. The high resistance of FLA cysts to disinfection methods is a trojan horse for public health insofar as they colonize water systems and allow the survival of intracellular microorganisms resistant to FLA. The results of this work advance current knowledge of the FLA population.