Stable carbon (C-13) and nitrogen isotopes (N-15) are useful tools in determining the presence of agricultural influences in freshwater ecosystems. Here we examined delta N-15 and delta C-13 signatures in nitrate, fish, and mussel tissues, from rivers in Southern Ontario, Canada, that vary in their catchment proportion of agriculture land use, nutrients and organic matter quality. We found comparatively N-15-enriched delta N-15 values in animal tissues and dissolved nitrates, relative to expected values characterized by natural sources. We also observed a strong, positive correlation between riparian agriculture percentages and delta N-15 values in animal tissues and nitrates, indicating a significant influence of agricultural land use and the probable dominance of organic fertilizer and manure inputs in particular. The use of a N-15-based equation for the estimation of fish trophic position confirmed dietary analyses is showing all fish species to be tertiary consumers, with a relatively consistent N-15-enrichment in animal tissues between trophic levels. This indicates that variability in N-15-trophic fractionation is minor, and that fish and mussel tissue delta N-15 values are largely representative of source nitrogen. However, the trophic fractionation value varied from accepted literature values, suggesting strong influences from either local environmental conditions or dietary variation. The delta C-13 datasets did not correlate with riparian agriculture, and animal delta C-13 signatures in their tissues are consistent with terrestrial C3 vegetation, suggesting the dominance of allochthonous DOC sources. We found that changes in water chemistry and dissolved organic matter quality brought about by agricultural inputs were clearly expressed in the delta N-15 signatures of animal tissues from all trophic levels. As such, this study confirmed the source of anthropogenic nitrogen in the studied watersheds, and demonstrated that this agriculturally-derived nitrogen could be traced with delta N-15 signatures through successive trophic levels in local aquatic food webs. The delta C-13 data was less diagnostic of local agriculture, due to the more complex interplay of carbon cycling and environmental conditions.