Contamination of Enterocytozoon bieneusi Desportes, Charpentier, Galian, Bernard, Cochand-Priollet, Laverne, Ravisse, et Modigliani, 1985 in water sources may cause outbreaks of microsporidiosis. To examine the occurrence of E. bieneusi, 108 raw wastewater samples were collected from three wastewater treated plants in Zhengzhou, China. In total, 46 samples were PCR positive for E. bieneusi. A total of 15 ITS genotypes was identified, including ten known genotypes (D, BEB6, I, J, PigEbIX, PigEBITS5, EbpA, Peru6, Peru8, Type IV) and five novel genotypes (HNWW1, HNWW2, HNWW3, HNWW4, HNWW5). Nine genotypes belonged to a known zoonotic group (group 1) and the other genotypes belonged to potential zoonotic group (group 2). Most of the genotypes had been identified in wildlife or domestic animals in former reports in Zhengzhou. The occurrence of E. bieneusi in wastewater was probably related to the rainfall day before sampling. Of 36 sampling days, 20 days had rainfall on the previous day and 16 days had none. As many as 43 of 60 samples were found to be E. bieneusi-positive in the 20 days which had rainfall on the previous day. Only three of 48 samples were found to be E. bieneusi-positive in the 16 days without rainfall the day before. The significant difference of the occurrence of E. bieneusi was observed between wet days and dry days by t-test (43/60 vs 3/48, p < 0.01). This indicates that the occurrence of E. bieneusi in wastewater in Zhengzhou mainly originated from animals and was probably related to rainfall the day before sample collection. Given the zoonotic genotypes detected in wastewater, animal faeces should be treated appropriately before being drained into the water source.