Information on the contribution of nitrogen (N) cycling processes in bed sediments to river nutrient fluxes in large northern latitude river systems is limited. This study examined the relationship between N-cycling processes in bed sediments and N speciation and loading in the Yukon River near its mouth at the Bering Sea. We conducted laboratory bioassays to measure N-cycling processes in sediment samples collected over distinct water cycle seasons. In conjunction, the microbial community composition in the bed sediments using genes involved in N-cycling (narG, napA, nosZ and amoA) and 16S rRNA gene pyrosequences was examined. Temporal variation was observed in net N mineralization, nitrate uptake and denitrification rate potentials and correlated strongly with sediment carbon (C) and extractable N content and microbial community composition rather than with river water nutrient concentrations. The C content of the bed sediment was notably impacted by the spring flood, ranging from 1.1% in the midst of an ice-jam to 0.1% immediately after ice-out, suggesting a build-up of organic material (OM) prior to scouring of the bed sediments during ice break-up. The dominant members of the microbial community that explained differences in N processing rates belonged to the genera Crenothrix, Flavobacterium and the family of Comamonadaceae. Our results suggest that biogeochemical processing rates in the bed sediments appear to be more coupled to hydrology, nutrient availability in the sediments and microbial community composition rather than river nutrient concentrations at Pilot Station.