Escherichia coli O157:H7 and Salmonella are pathogenic microorganisms that can cause severe gastrointestinal illness in humans. These pathogens may be transmitted in a variety of ways, including food and water. The presence of Salmonella and E. coli O157:H7 in surface waters constitutes a potential threat to human health when used for either drinking or recreation. As with most waterborne pathogens, Salmonella and E. coli O157:H7 are difficult to detect and enumerate with accuracy in surface waters due to methodological limitations. The aim of this study was to develop a protocol for the detection of Salmonella spp., E. coli O157:H7 and E. coli virulence genes (stx (1), stx (2) and eae) in water using a single enrichment step and PCR. In spiked water samples, PCR results showed high sensitivity (<3 CFU/L) for both microorganisms. The protocol developed in this study has been applied in different surface waters in association with microbiological and physical analysis. The frequency of PCR positive samples was 33% for Salmonella and 2% for E. coli O157:H7 producing intimin (eae) and Shiga-like toxin I (stx (1)). Moreover, the finding of amplicons corresponding to eae and stx (1) genes in the absence of E. coli O157:H7 suggested the possible presence of other pathogenic bacteria that carry these genes (e.g. EHEC, Shigella strains). The results obtained showed that the developed protocol could be applied as a routine analysis of surface water for the evaluation of microbiological risks.