Stable carbon (13C) and nitrogen isotopes (15N) are useful tools in determining the presence of agricultural influences in freshwater ecosystems. Here we examined δ15N and δ13C 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 15N-enriched δ15N 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 δ15N 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 15N-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 15N-enrichment in animal tissues between trophic levels. This indicates that variability in 15N-trophic fractionation is minor, and that fish and mussel tissue δ15N 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 δ13C datasets did not correlate with riparian agriculture, and animal δ13C 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 δ15N 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 δ15N signatures through successive trophic levels in local aquatic food webs. The δ13C data was less diagnostic of local agriculture, due to the more complex interplay of carbon cycling and environmental conditions.