Recent water related outbreaks of shiga toxin producing E. coli O157 have resulted in increased attention of the water industry to this potentially deadly pathogen. Current methods to detect E. coli O157 and its virulence genes are laborious and time-consuming. Specificity, sensitivity and simple use of a real-time PCR method makes it an attractive alternative for the detection of STEC E. coli O157. This study describes the development and application of real-time PCR methods for the detection of E. coli O157, shiga toxin genes (Stx1 and Stx2) and E. coli. The specificity of the methods was confirmed by performing colony-PCR assays on characterized bacterial isolates, demonstrating the applicability of these assays as rapid tests to confirm the presence of E. coli or E. coli O157 colonies on culture plates. Sensitive culture-PCR methods were developed by combining culture enrichment with real-time PCR detection. This rapid method allowed detection of low concentrations of E. coli O157 in the presence of high concentrations of non-O157-E. coli (1:104). Culture-PCR methods were applied to 27 surface water and 4 wastewater samples. E. coli O157 and both Stx genes were detected in two wastewater samples, whereas only E. coli O157 was detected in two surface water samples. Culture-PCR methods were not influenced by matrix effects and also enabled quantitative (MPN) detection of E. coli in these samples.