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Background: Acanthamoeba is an opportunistic amphizoic protozoan found in different wa­ter sources including swimming pool as well as in sewage. The aim of this study was to in­vestigate the prevalence of Acanthamoeba in tap-water samples in Iran. Method: In this descriptive cross-sectional study, 94 samples of cold and warm tap-water were collected from different wards of hospitals in 13 cities of Iran in 2007-2008. Free resi­dual chlorine, pH, and temperature of samples were measured. After filtration through multi­pore nylon membrane, samples were cultured on non-nutrient agar. Then we investigated ex­istence of Acanthamoeba by reverse contrast phase microscope. Results: Acanthamoeba was found in 45 samples (48%). Thirty-four and 11 positive samples were collected from cold and warm tap water, respectively. The samples belonged to the cate­gory of 20-30 °C temperature with 0-2 ppm free residual chlorine and pH 6-7.4 showed the most coincidence to the positive cases. The greatest proportion of positive samples was ob­tained from Mashhad hospitals, while all samples collected from Arak and Semnan hospitals were negative. Conclusion: considering the results of this study and the pathogenic role of this protozoan on pa­tients with immunodeficiency, as well as capability of this microorganism in carrying other pathogens such as Legionella, further studies are needed. What is more important, potable water in hospitals should follow the procedure of treatment and sanitation, in order to prevent the relevant nosocomial infections.