In aquatic ecosystems, the biomass, activity and composition of microorganisms are determined to a large extent by local and regional environmental conditions. While karst aquifers are an important source for drinking water, the ecology of microbial communities in karst waters has hardly been studied. This study examined the regional variations and seasonal dynamics of microbial communities in pristine karst spring waters of Slovenia (Central Europe). Fifteen springs distributed across 5 eco-regions exhibiting a strong altitudinal gradient were sampled 4 times a year. Evaluation of the microbial communities included quantification of prokaryotic biomass via total cell counts and microbial activity estimated via measurements of electron transport system activity. The taxonomic structure of the bacterial communities was analysed by terminal restriction fragment length polymorphism fingerprinting. Biological measures were complemented by a set of physico-chemical parameters, including dissolved organic carbon, nutrients, major ions, temperature, electrical conductivity, pH, and dissolved oxygen. Bacterial community structure differed significantly with seasons and eco-regions, with the latter causing greater variation. While the overall power of the environmental variable tested was a moderate factor (15%) in explaining the variability in microbial community structure, catchment altitude was a key determinant. Prokaryotic cell density and microbial activity in spring water decreased with an increase in catchment altitude and were significantly positively correlated. For a better understanding of karst ecosystems and the ecosystem service of water purification, future investigation should address karst microbial communities at a higher phylogenetic and functional resolution.