Bacterial communities in human-impacted rivers and streams are exposed to multiple anthropogenic contaminants, which can eventually lead to biodiversity loss and function. The Wonderfonteinspruit catchment area is impacted by operational and abandoned gold mines, farms, and formal and informal settlements. In this study, we used 16S rRNA gene high-throughput sequencing to characterize bacterial communities in the lower Wonderfonteinspruit and their response to various contaminant sources. The results showed that composition and structure of bacterial communities differed significantly (P<0.05) between less (downstream) and more (upstream) polluted sites. The taxonomic and functional gene dissimilarities significantly correlated with each other, while downstream sites had more distinct functional genes. The relative abundance of Proteobacteria, Bacteroidetes and Actinobacteria was higher at upstream sites, while Acidobacteria, Cyanobacteria, Firmicutes and Verrucomicrobia were prominent at downstream sites. In addition, upstream sites were rich in genera pathogenic and/or potentially pathogenic to humans. Multivariate and correlation analyses suggest that bacterial diversity was significantly (P<0.05) impacted by pH and heavy metals (cobalt, arsenic, chromium, nickel and uranium). A significant fraction (~14%) of the compositional variation was explained by a combination of anthropogenic inputs, of which mining (~6%) was the main contributor to bacterial community variation. Network analysis indicated that bacterial communities had non-random inter- and intra-phyla associations and that the main taxa showed both positive and negative linkages to environmental parameters. Our results suggest that species sorting, due to environmental parameters, was the main process that structured bacterial communities. Furthermore, upstream sites had higher relative abundances of genes involved in xenobiotic degradation, suggesting stronger removal of polycyclic aromatic hydrocarbons and other organic compounds. This study provides insights into the influences of anthropogenic land use on bacterial community structure and functions in the lower Wonderfonteinspruit.