Recent work suggests that microbial community composition in high-elevation lakes is significantly influenced by microbes entering from upstream terrestrial and aquatic habitats. To test this idea, we conducted 18S and 16S rDNA surveys of microbial communities in a high-alpine lake in the Colorado Rocky Mountains. We compared the microbial community of the lake to water entering the lake and to uphill soils that drain into the lake. Utilizing hydrological and abiotic data, we identified potential factors controlling microbial diversity and community composition. Results show a diverse community entering the lake at the inlet with a strong resemblance to uphill terrestrial and aquatic communities. In contrast, the lake communities (water column and outlet) showed significantly lower diversity and were significantly different from the inlet communities. Assumptions of neutral community assembly poorly predicted community differences between the inlet and lake, whereas “variable selection” and “dispersal limitation” were predicted to dominate. Similarly, the lake communities were correlated with discharge rate, indicating that longer hydraulic residence times limit dispersal, allowing selective pressures within the lake to structure communities. Sulfate and inorganic nitrogen and phosphorus concentrations correlated with community composition, indicating “bottom up” controls on lake community assembly. Furthermore, bacterial community composition was correlated with both zooplankton density and eukaryotic community composition, indicating biotic controls such as “top-down” interactions also contribute to community assembly in the lake. Taken together, these community analyses suggest that deterministic biotic and abiotic selection within the lake coupled with dispersal limitation structures the microbial communities in Green Lake 4.