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Lake Erie is the most at risk of the Great Lakes for degraded water quality due to non-point source pollution caused by agricultural activities in the lake's watershed. The extent and temporal patterns of nutrient loading from these agricultural activities is influenced by the timing of agronomic events, precipitation events, and water flow through areas of natural filtration within the watershed. Downstream impacts of these nutrient loading events may be moderated by the co-loading of functionally relevant biogeochemical cycling microbial communities from agricultural soils. This study quantified loading patterns of these communities from tile drain sources, assessed whether functional communities from agricultural sources influenced downstream microbial functionality, and investigated how distance from agricultural sources, storm events, and areas of natural filtration altered nutrient cycling and nutrient fluxes in aquatic and sediment environments. Water and sediment samples were collected in the Wigle Creek watershed in Ontario, from tile drains through to Lake Erie, from May to November 2021, and microbial nitrogen (N) and phosphorous (P) cycling capacity (quantitative PCR), and nutrient levels were evaluated. Results showed that N and P functional groups were co-loaded with nutrients, with increased loading occurring during storm events and during agricultural activities including fertilization and harvest. Overall functional capacity in the aquatic environment decreased with distance from the agricultural sources and as water transited through natural filtration areas. In contrast, the sediment environment was more resilient to both agricultural disturbances and abiotic factors. This study expands our understanding of when and where different stages of N and P cycling occurs in agriculturally impacted watersheds, and identifies both seasons and regions to target with nutrient mitigation strategies.