Dimba Cave is a large array of natural galleries in limestone mountains of the Democratic Republic of the Congo that contains highly valued pre-historic archaeological artifacts. The cave attracts a high number of tourists every year and is used by local populations as a water supply source. The main objective of the research undertaken in Dimba Cave consisted of assessing the quality of water and sediments from Dimba Cave ponds through evaluating contamination by heavy metals (15 elements analyzed, including As, Cd, Pb, and Hg) and by microbial populations (including Escherichia coli and total coliforms) in order to estimate the ecotoxicological risk to humans and to non-human biota. All water samples collected in the cave ponds showed very high metal concentrations exceeding the internationally recommended limits for drinking water, particularly for Cr, Mn, As, Pb, and Hg. Most sediment samples from cave ponds also displayed high heavy metal concentrations. The calculated pollution parameters, such as the enrichment factor (EF), and ecological risk parameters, such as the ecological risk index (Eri), indicated that the sediment may be toxic to aquatic biota. Furthermore, the microbiological analysis of pond waters indicated a widespread contamination with bacteria such as Escherichia coli, Enterococcus spp., total coliforms, and Pseudomonas spp., probably from anthropogenic and/or animal sources. Therefore, the consumption of Dimba Cave water as a drinking water represents a threat to public health. Urgent management measures should be enforced to protect public health and the cave ecosystem.