© 2015 Titilawo et al. Background: Diarrhoeal diseases are attributable to unsafe water stemming from improper sanitation and hygiene and are reportedly responsible for extensive morbidity and mortality particularly among children in developed and developing countries. Methods: Water samples from selected rivers in Osun State, South-Western Nigeria were collected and analyzed using standard procedures. Escherichia coli isolates (n=300) were screened for 10 virulence genes using polymerase chain reaction for pathotyping. Results: While the virulence gene (VG) lt for enterotoxigenic E. coli had the highest prevalence of 45 %, the enteropathogenic E. coli genes eae and bfp were detected in 6 and 4 % of the isolates respectively. The VGs stx1 and stx2 specific for the enterohemorrhagic E. coli pathotypes were detected in 7 and 1 % of the isolates respectively. Also, the VG eagg harboured by enteroaggregative pathotype and diffusely-adherent E. coli VG daaE were detected in 2 and 4 % of the isolates respectively and enteroinvasive E. coli VG ipaH was not detected. In addition, the VGs papC for uropathogenic and ibeA for neonatal meningitis were frequently detected in 19 and 3 % of isolates respectively. Conclusions: These findings reveal the presence of diarrhoeagenic and non-diarrhoeagenic E. coli in the selected rivers and a potential public health risk as the rivers are important resources for domestic, recreational and livelihood usage by their host communities.