Acanthamoeba species are free-living amoebae having worldwide distribution. These amoebae can cause granulomatous amoebic encephalitis and amoebic keratitis in humans. They can produce proteases that are considered virulence factors. Acanthamoeba can also harbor pathogenic bacteria, fungi, and viruses. The objective of this study is to evaluate the presence of Acanthamoeba in the environment of Helwan University, Egypt. Six types of samples (tap water, irrigation water, wastewater, swabs from surfaces, soil, and air) were collected, processed, and cultured on non-nutrient agar medium. Positive plates for Acanthamoeba were subcultured, purified and amoebae were identified morphologically and confirmed by PCR using Acanthamoeba genus-specific primers. Obtained results declared that members of genus Acanthamoeba were detected in 91.7, 83.3, 54.2, 45.8, 12.5 and 12.5% of irrigation water, soil, swabs, wastewater, tap water, and air samples, respectively. The morphologically identified Acanthamoeba species proved to be related to genus Acanthamoeba when tested by PCR. Statistically, the sampling source had a strong significant correlation with the prevalence of Acanthamoeba. The highest appearance of Acanthamoeba was recorded in the spring season for samples from irrigation water, soil, and swabs from surfaces. In conclusion, the high prevalence of Acanthamoeba species in irrigation water and soil exert public health hazards to students and workers in Helwan University.