Multiple factors operating across different spatial and temporal scales affect beta-diversity—the variation in community composition among sites. Disentangling the relative influence of co-occurring ecological drivers over broad biogeographic gradients and time is critical to developing mechanistic understandings of community responses to natural environmental heterogeneity as well as predicting the effects of anthropogenic change. We partitioned taxonomic beta-diversity in phytoplankton communities across 75 north-temperate lakes and reservoirs in Alberta, Canada, using data-driven, spatially-constrained null models to differentiate between spatially-structured, spatially-independent, and spuriously correlated associations with a suite of biologically relevant environmental variables. Beta-diversity was largely independent of space, indicating spatial processes (e.g. dispersal limitation) likely play a minor role in structuring freshwater phytoplankton communities at the regional scale. Our findings also identified seasonal differences in the importance of environmental factors, suggesting a general shift towards greater relevance of local, in-lake (e.g. nutrients and Secchi depth) over regional, atmospheric and catchment-level (e.g. monthly solar radiation and grassland coverage) drivers as the open-water growing season progressed. Several local and regional variables explained taxonomic variation jointly, reflecting climatic and land-use linkages (e.g. air temperature and water column stability or pastureland and nutrient enrichment) that underscore the importance of understanding how phytoplankton communities integrate—and may serve as sentinels of—broader anthropogenic changes. We also discovered similar community composition in natural and constructed water bodies, demonstrating rapid filtering of regional species to match local environmental conditions in reservoirs comparable to those in natural habitats. Finally, certain factors related to human-footprint (e.g. cropland development) explained the composition of bloom-forming and/or toxic cyanobacteria more than the overall phytoplankton community, suggesting their heightened importance to integrated watershed management.