The aim of this study was to evaluate the spatial and temporal variation of microbiological parameters of the Lower Vaal River, with emphasis on the Staphylococci population. River water concentrations of Staphylococci, heterotrophic, total coliforms, fecal coliforms and Streptococci groups of sanitary indicator bacteria were monitored. Results indicated significant contamination from agricultural land use inputs and municipal waste applications. Significant temporal and spatial variation was observed in response to the varying river discharge. Higher microbial concentrations were detected during high river discharge whereas during low river discharge both low and high microbial concentrations were detected. The varying responses associated with river discharge helped to identify the importance of different sources of contamination in the catchment and the mechanisms transferring them. The overall impact of contamination on the water quality of the Vaal River could have potentially serious public health risks but also provide valuable data for integrated water resource management.