Groundwater flow influenced epibenthic algal biomass and N:P ratios at a seepage lake (Sparkling Lake, Wisconsin). During seasonal studies, biomass and seepage flux were positively associated (r = 0.453; P < 0.001). Pore‐water soluble reactive phosphorus (SRP) concentrations (29.2–110.7 µg PO, liter−1), SRP fluxes, and algal biomass were significantly higher at high groundwater discharge sites than at low flow sites (< 10.0 to 27.7 µg PO4 liter−1). Pore‐water ammonia (NH4+) concentrations were significantly lower at high groundwater discharge sites (<10.0 to 566.0 µg NH4+ liter−1) than at low groundwater discharge and recharge sites (61.4–1464.9 µg NH3 liter−1). The coupling between pore‐water nutrient concentrations and local groundwater flow dynamics suggests a mechanism for the observed spatial patterns in biomass. In situ experimental chambers evaluated coupling between epibenthic algal biomass, N:P ratios, and groundwater flow patterns. Biomass responded rapidly in chambers, reaching ambient levels within 1.5 months of initiation. Free‐flow chambers in discharge regions had consistently higher soluble reactive phosphorus (SRP), NO3‐NO2, and O2 concentrations, higher phosphate and nitrate‐nitrite fluxes, higher algal biomass, and lower N:P ratios in the developing mat. Free‐flow chambers in recharge regions had high ammonia (NH3) concentrations, lower algal biomass, and higher N:P ratios. These results confirm that groundwater‐related nutrient fluxes influence the local physicochemical environment and affect epibenthic algal biomass.