Water column samples from the island of Mallorca, Spain were collected from one site in Cova des Pas de Vallgornera (Vallgornera) and three sites (Llac Martel, Llac Negre, and Llac de les Delícies) in Coves del Drac (Drac). Vallgornera is located on the southern coast of Mallorca approximately 57 km southwest of Coves del Drac. Drac is Europe's most visited tourist cave, whereas Vallgornera is closed to the public. Water samples were analyzed for water chemistry using spectrophotometric methods, by quantitative PCR for estimated total abundance of microbial communities, and by length heterogeneity PCR for species richness and relative species abundance of Archaea, Bacteria, and microbial eukaryotes. Estimated total abundance was multiplied by relative species abundance to determine the absolute species abundance. All sites were compared to determine spatial distributions of the microbial communities and to determine water column physical and chemical gradients. Water quality and community structure data indicate that both Drac Delícies and Drac Negre have distinct biogeochemical gradients. These sites have communities that are similar to Vallgornera but distinct from Drac Martel, only a few hundred meters away. Drac Martel is accessible to the general public and had the most dissimilar microbial community of all the sites. Similarities among communities at sites in Drac and Vallgornera suggest that these two spatially separated systems are operating under similar ecological constraints.