Aquifer systems are composed of water flowing from surface recharge areas, to the subsurface and back to the surface in discharge regions. Groundwater habitats harbor a large microbial biomass and diversity, potentially contributing to surface aquatic ecosystems. Although this contribution has been widely studied in marine environments, very little is known about the connection between underground and surface microbial communities in freshwater settings. Therefore, in this study, we used amplicon sequencing to analyze the archaeal, bacterial, and eukaryotic community diversity and structure in groundwater and surface water samples, spanning the vast regions of the Laurentides and Lanaudières in the Quebec province (Canada). Our results show significant differences between subsurface and surface taxa; with more fungi, Amoebozoa, and chemolithoautotrophic prokaryotes involved in nitrogen-, sulfur-, and iron-cycling dominating the underground samples; while algae, ciliates, methanogens, and Actinobacteria dominate the surface discharge waters. Microbial source tracking suggested that only a small portion of the microbial communities in the groundwater contributed to the surface discharge communities. However, many taxa were shared between both habitats, with a large range of functional diversity, likely explaining their survival in both subsurface and surface water ecosystems.