Introduction: Free-living amoebas are opportunistic amoebae that usually live freely in various environmental conditions, including warm waters and even in water supply network pipes and reservoirs connected to these waters. In addition to living freely, these protozoa are able to attack the host's body when they enter a human or animal body. Therefore, it is necessary to identify their presence in water resources. So, the aim of this study was to identify free-living amoebas isolated from water reservoirs of hospitals in southwest Iran. Methods: A total of 80 water samples were isolated from the hospitals of Ahvaz city, southwest Iran, and then their physical and chemical parameters were measured. The samples were then fed to a non-food agarculture medium and stained using the Giemsa-Wright staining. Finally, the samples were identified by the PCR molecular method. Results: The mean pH and turbidity values were 7.57 ± 0.03 and 3.31 ± 0.26 nephelometric turbidity unit (NTU), respectively. The mean residual chlorine and electrical conductivity were 0.91 ± 0.02 and 1122.39 ± 24.31, respectively. In addition, 9 (11.25%) and 3 (3.75%) samples were contaminated with Acanthamoeba spp. and Naegleria spp., respectively. However, no positive cases of Balamuthia spp. infection were observed. Moreover, two samples were co-infected with Acanthamoeba spp. and Naegleria spp. Conclusion: Due to the existence of free-living amoebas in water storage tanks at hospitals, it is necessary to prevent possible contamination with these amoebas and infectious agents by using new methods of disinfection and purification of water resources.