Aims: There is limited data on the occurrence of E. Coli O157: H7 in water. Therefore, this study aims to detect E. Coli O157: H7 in the Water Treatment Plant (WTP). Materials and Methods: This cross-sectional study was conducted in Isfahan WTP, central of Iran. Immunological methods were implemented with anti-serum kits and the molecular method of reverse transcription-polymerase chain reaction (RT-PCR) was used to detect E. Coli O157: H7 in eight locations of the WTP; the sludge of the sedimentation basin and filter backwash water were also monitored. The survival of E. Coli O157: H7 in the sludge samples of the sedimentation basin was indicated by the formation of agglutination particles using the immunological method, and through indicator probes using the RT-PCR method. Results: E. Coli O157: H7 was not detected in the water samples from the WTP units. The removal percent of total coliforms (TC), fecal coliforms (FC), and Heterotrophic Plate Count (HPC), respectively, were as follows: 59.5, 49, and 54.8% in the sedimentation basin; 66, 45.8, and 57% in the ozonation system; 98.8, 98, and 78.8% in the filtration system; and 96, 100, 91% in the disinfection system. Conclusions: This study revealed the existence of the pathogenic coliform of E. Coli O157: H7 in the sludge of the sedimentation basin. The absence of E. Coli O157: H7 in the finished water indicated that the WTP units were able to eliminate these pathogenic bacteria before reaching the final units of the plant, including the filtration and disinfection systems.