The protistan genus Acanthamoeba is a free-living amoeba existing in various environments. Within this protistan genus, there are some species recognized as potential human pathogens, which may cause granulomatous amoebic encephalitis, Acanthamoeba keratitis and chronic granulomatous lesions of the skin. In this study, 211 water samples were collected from two watersheds in southern Taiwan. We detected Acanthamoeba based on the PCR amplification with a genus-specific primer pair and investigation of Acanthamoeba in Puzih River and Kaoping River in southern Taiwan. Acanthamoeba species were detected in 34 (16.1%) samples. The presence or absence of Acanthamoeba within the water samples showed significant difference with the levels of water temperature and total coliforms. The most frequently identified Acanthamoeba genotype was T4 (n = 19), followed by T5 (n = 8), and then T15 (n = 3). Genotype T6, T7/T8, T11 and T12 were all detected once. Genotype T4, T5, T6, T11 and T15 of Acanthamoeba are responsible for Acanthamoeba keratitis and should be considered a potential health threat associated with human activities in environmental surface water watersheds.