Land-use practices can deteriorate the health of aquatic ecosystems in lower-order streams by increasing the availability of nutrients to periphytic algae. The magnitude of eutrophication likely depends in part on the nature of nutrient limitation and other local environmental factors. We used nutrient diffusing substrates and assessments of taxonomically diagnostic pigments to experimentally identify the drivers of nutrient limitation of periphytic algal assemblages across 30 streams which collectively spanned a gradient of agricultural land-use intensity (cropland, pasture, and nutrients) across Boreal Forest, Grassland, and Parkland ecoregions in Alberta, Canada. We identified N as the primary limiting factor of total algal biomass accrual. Overall, bacillariophytes made up most of the total algal biomass response to N. Dissolved inorganic N to total P ratios in the water column were consistently low, and P amendment altered assemblage composition by stimulating chlorophytes over bacillariophytes. Differences in soil types, precipitation, runoff, and leaching potential within and across ecoregions did not alter the algal responses to the experimental nutrient amendments. The effect of N-limiting conditions observed across most streams had greater relative importance than other potential abiotic factors (e.g., light) on algal growth. Whereas a historical focus on P management remains important, our findings highlight the need to increase the attention and focus on N in watershed management plans during the summer months to mitigate eutrophication of agricultural streams.