Escherichia coli (E. coli) consists of commensal (ComEC) and diarrhoeagenic (DEC) groups. ComEC are detected using traditional culture methods. Conformational steps are performed after culturing if it is required to test for the presence of DEC, increasing cost and time in obtaining the results. The aim of this study was to develop a single-step multiplex polymerase chain reaction (m-PCR) that can simultaneously amplify genes associated with DEC and ComEC, with the inclusion of controls to monitor inhibition. A total of 701 samples, taken from clinical and environmental water sources in South Africa, were analysed with the optimised m-PCR which targeted the eaeA, stx1, stx2, lt, st, ial, eagg, astA and bfp virulence genes. The mdh and gapdh genes were included as an internal and external control, respectively. The presence of the external control gapdh gene in all samples excluded any possible PCR inhibition. The internal control mdh gene was detected in 100 % of the environmental and 85 % of the clinical isolates, confirming the classification of isolates as E. coli PCR positive samples. All DEC types were detected in varying degrees from the mdh positive environmental and clinical isolates. Important gene code combinations were detected for clinical isolates of 0.4 % lt and eagg. However, 2.3 % of eaeA and ial, and 8.7 % of eaeA and eagg were reported for environmental water samples. The E. coli astA toxin was detected as positive at 35 and 17 % in environmental isolates and clinical isolates, respectively. Interestingly, 25 % of the E. coli astA toxin detected in environmental isolates and 17 % in clinical isolates did not contain any of the other virulence genes tested. In conclusion, the optimised single-step 11-gene m-PCR reactions could be successfully used for the identification of pathogenic and non-pathogenic E. coli types. The m-PCR was also successful in showing monitoring for PCR inhibition to ensure correct reporting of the results.