Bacteria are essential components of natural environments. They contribute to ecosystem functioning through roles as mutualists and pathogens for larger species, and as key components of food webs and nutrient cycles. Bacterial communities respond to environmental disturbances, and the tracking of these communities across space and time may serve as indicators of ecosystem health in areas of conservation concern. Recent advances in DNA sequencing of environmental samples allow for rapid and culture-free characterization of bacterial communities. Here we conduct the first metabarcoding survey of bacterial diversity in the waterholes of the Kruger National Park, South Africa. We show that eDNA can be amplified from waterholes and find strongly structured microbial communities, likely reflecting local abiotic conditions, animal ecology, and anthropogenic disturbance. Over timescales from days to weeks we find increased turnover in community composition, indicating bacteria may represent host-associated taxa of large vertebrates visiting the waterholes. Through taxonomic annotation we also identify pathogenic taxa, demonstrating the utility of eDNA metabarcoding for surveillance of infectious diseases. These samples serve as a baseline survey of bacterial diversity in the Kruger National Park, and in the future, spatially distinct microbial communities may be used as markers of ecosystem disturbance, or biotic homogenization across the park.