Point- and non-point source nutrients are likely to have different ecological impacts in receiving waters, due to differences in the concentration and proportions of nutrient fractions. However, the direct comparison of their ecological impacts in receiving waters has barely been quantified. We undertook algal bioassays with algal communities from river sites and showed that there was a photosynthetic yield (Fv/Fm) response to nutrient enrichment when river nutrient concentrations were relatively low, but not at higher nutrient concentrations. To combat this variability in the photosynthetic state of algae, we developed a standardized algal bioassay (3-day), using a cultured species of algae which was starved of nitrogen, to compare the photosynthetic response to three nitrogen sources: treated wastewater, aquaculture farm discharges, and soil erosion-derived nutrient sources. This study showed that the nutrient parameter that had the highest correlation with algal photosynthetic response was total dissolved nitrogen (TDN), i.e., the sum of dissolved inorganic and organic nitrogen, rather than dissolved inorganic nitrogen alone. This was true across all three nutrient sources (R2 = 0.58–0.78). Additionally, the same concentrations of TDN from soil erosion-derived and aquaculture samples resulted in a significantly higher algal photosynthetic response, compared to the treated wastewater. This indicates that TDN from soils and aquaculture farms was significantly more bioavailable to the cultured algae than treated wastewater. When a range of parameters were correlated with algal responses, organic and inorganic nutrients, and organic carbon were the parameters that had the highest explanatory power for soil erosion-derived and aquaculture samples (R2 = 0.75–0.87). The importance of organic compounds in these equations points to the potential of microbial transformation of organic nutrients into more bioavailable forms during the 3-day bioassay. This highlights the need to understand the relationship between algal and microbial communities in natural systems for nutrient source impact assessment. This study provides an improved understanding and metrics for comparing the algal growth response to different nutrient sources.