The landscape position of a lake relative to local and regional groundwater flow patterns has important ecosystem consequences. The local hydraulic gradient sets up seepage regimes, influences hydrologic and nutrient budgets, and alters site stoichiometry (molar N:P). However, little is known about the biotic (ecological) consequences of groundwater–lake coupling. We investigated the relationship between groundwater flux (resource supply rates), resource supply ratios, and species patterns of benthic algae in Sparkling Lake, Wisconsin, USA, a north-temperate flow-through seepage lake. Nutrient fluxes of seepage waters influenced both local benthic algal biomass and species composition. Sites of high groundwater discharge (HGD) were characterized by low N:P flux, high algal biovolume, and low species richness and diversity. In contrast, low groundwater discharge (LGD) and low groundwater recharge (LGR) sites had high N:P, low algal biovolume, and high richness and diversity. HGD sites were dominated by diatoms and cyanobacteria characteristic of high P, whereas LGD and LGR sites were characterized by taxa associated with low P. Many species were unique to the local flow regimes, increasing overall benthic algal species richness in Sparkling Lake.