South Africa’s freshwater quality and quantity is declining and consequently impacting on the ecological health of these ecosystems, due to increased agricultural, urban and industrial developments. The River Health Programme (RHP) was designed for monitoring and assessing the ecological health of freshwater ecosystems in South Africa, in order to effectively manage these aquatic resources. The RHP utilises biological indicators such as in-stream biota as a structured and sensitive tool for assessing ecosystem health. Although the RHP has been widely implemented across South Africa, no attempts have been made to explore microbial ecology as a tool that could be included as one of the RHP indices. This study used selected microbial responses and water physico-chemical parameters to assess the current water quality status of the Buffalo River. This study showed that water quality impairments compounded in the urban regions of King William’s Town and Zwelitsha and also downstream of the Bridle Drift Dam. The results also showed that the lower and the upper catchments of the Buffalo River were not significantly different in terms of water physico-chemistry and microbiology, as indicated by low stress levels of an NMDS plot. Though similarities were recorded between impacted and reference sites, the results strongly showed that known impacted sites recorded the poorest water physico-chemistry, including the Yellowwoods River. However, the Laing Dam provided a buffer effect on contributions of the Yellowwoods River into the Buffalo River. Multivariate analysis showed that microbial cell counts were not influenced by water physico-chemical changes, whilst microbial activity from the water and biofilm habitats showed significant correlation levels to water physico-chemical changes. This study demonstrated that further investigations towards exploitation of microbial activity responses to water physico-chemical quality changes should be channelled towards the development of microbiological assessment index for inclusion in the RHP.