The prevalence of adenovirus (AdV), rotaviruses (RV) and enteroviruses (EV) in Umgeni River waters of Durban, South Africa was assessed qualitatively and quantitatively during April 2011 to January 2012 using polymerase chain reaction (PCR)/reverse transcription-polymerase chain reaction (RT-PCR), nested PCR and quantitative PCR (qPCR), as well as nested integrated cell culture PCR (nested ICC-PCR). The phylogenetic analysis of the adenovirus and enterovirus amplicons was also performed. The nested PCR results effectively detected the presence of AdV and EV in all water samples. The results of qPCR demonstrated that higher populations of EV and of AdV were widely found in the Umgeni River. Rotavirus could only be detected in the upper Umgeni River, mainly during drier seasons. Nested ICC-PCR further confirmed the presence of infectious AdV and EV particles in 100% of water samples using various cell lines. The present study identifies potential viral hazards of Umgeni River water for domestic water supply and recreational activities.