The terrestrial subsurface harbors unique microbial communities that play important biogeochemical roles and allows studying a yet unknown fraction of the Earth’s biodiversity. The Saint-Leonard cave in Montreal city (Canada) is of glaciotectonic origin. Its speleogenesis traces back to the withdrawal of the Laurentide Ice Sheet, 13 000 years ago, during which the moving glacier dislocated the sedimentary rock layers. Our study is the first to investigate the microbial communities of the cave. By using amplicon sequencing, we analyzed the taxonomic diversity and composition of bacterial, archaeal and eukaryotes communities living in the groundwater (0.1µm- and 0.2µm-filtered water), in the sediments and in surface soils. We identified a microbial biodiversity typical of cave ecosystems. Communities were mainly shaped by habitat type and harbored taxa associated with a wide variety of lifestyles and of metabolic capacities. Although we found evidence of a geochemical connection between the above soils and the cave’s galleries, our results suggest that the community assembly dynamics are driven by habitat selection rather than dispersal. Furthermore, we found that the cave’s groundwater, in addition to being generally richer in microbial taxa than sediments, contained a considerable diversity of ultra-small bacteria and archaea.