Management efforts to reduce cyanobacterial harmful algal blooms (cHABs) in the Great Lakes have focused on decreasing tributary inputs of phosphorus (P). Recent research has indicated that reduction of both P and nitrogen (N) can lessen cHABs severity. Microbially mediated N cycling in streambed sediment may reduce N riverine loads, yet little is known about in-stream N processing rates in the Maumee River Basin, a major source of nutrients to Lake Erie. During summer of 2019 and 2021, we sampled streambed sediment to measure potential nitrification and denitrification rates using the acetylene block method at 78 sites throughout the Maumee River network. We used structural equation models to identify indirect and direct drivers of denitrification. Precipitation was greater in 2019, resulting in a 67 % increase in mean discharge, 41 % of farm fields to be fallow, and a 50 % reduction in fertilizer use. During summer field surveys, median stream-water nitrate concentrations were not different between 2019 and 2021. Median denitrification rates were 13.3 mg N/m2/h and 31.2 mg N/m2/h, respectively, indicating high potential to remove N. Nitrate concentrations and nitrification rates were strong direct drivers of denitrification, especially in 2019 when coupled nitrification–denitrification sustained denitrification. Nitrate concentrations varied with land use. Notably, nitrate concentrations increased with the area of fallow land, which may indicate the presence of a legacy N source. These findings indicate that promoting streambed denitrification could reduce N loads to Lake Erie, but legacy N currently stored in the system may mask N reduction efforts.