Understanding the mechanisms governing nitrate uptake in aquatic ecosystems is paramount in mitigating the impact of increased anthropogenic nitrogen loading on water quality. An experimental laboratory flume with agricultural sediment and carbon-amended sediment was used to evaluate the effect of turbulent fluid flow above rough sediment on oxygen uptake, nitrate uptake, and sediment bacterial gene abundances. The transport of dissolved oxygen to the sediment scaled with the friction velocity and sediment roughness height. Results demonstrated that nitrate uptake was mediated by turbulence levels, as quantified by friction velocity, above the sediment-water interface. Facilitated nitrate uptake for amended and unamended sediment occurred under midrange of friction velocities from 0.75 to 1.2 cm/s, which corresponds to Reynolds numbers from 2,010 to 5,200. High turbulence levels in the water column above the sediment with friction velocities larger than 1.40 cm/s or Reynolds numbers larger than 5,500, as well as low turbulence with friction velocities less than 0.65 cm/s or Reynolds numbers less than 1,400, were determined to minimize nitrate uptake by the sediment. Carbon-amended sediment had approximately 100 times greater nitrate uptake fluxes compared to field-collected unamended sediment. For unamended sediment experiments, gene abundances significantly increased over the course of the experiments for midrange friction velocities; increases were not observed in the low and high friction velocities. Gene abundances did not significantly increase in any experiments for carbon-amended sediment. Maintaining optimal turbulence levels could facilitate sustained nitrate uptake and reduce nitrogen loading to higher-order streams in aquatic ecosystems.