The effects of overlying flow conditions on periphyton structure and denitrification were measured in three laboratory mesocosms (120 cm long and 60 cm wide) under average velocities of 0.05, 0.5 and 5 cm/s. Periphyton was cultivated on polyethylene benthic nets overlaying a thin layer of sand. The mesocosms were operated continuously for four months, leading to prolific growth of periphyton on the benthic nets and in the underlying sediments. Periphyton structural characteristics were quantified in terms of algal/bacterial biomass, algal species composition, and microbial enumeration. Confocal microscopy was used to investigate the spatial organization of the periphyton. The denitrification potential of the microbial community in each mesocosm was evaluated using the acetylene inhibition method. Different benthic microbial communities developed under the three flow conditions, while the total microbial biomass accrual increased monotonically with increasing overlying velocity. Denitrification potential also increased with overlying velocity when evaluated on both a per unit area and per unit biomass basis. The periphytic community that developed under the fastest velocity and was characterized by the largest fractional biovolume of diatoms (predominantly Achnanthidium minutissimum), promoted establishment of a consortium of bacterial denitrifiers more physiologically active than those that developed under slower overlying velocities. These results demonstrate that hydrodynamic transport conditions play a key role in structuring benthic microbial communities, and the structural differences that develop under different flow conditions also regulate benthic microbial processing of substances delivered from the water column. Understanding the response of microbial communities to physical conditions is essential for evaluating nutrients dynamics, as well as for the development of management strategies aimed at mitigating effects of excess nitrogen by increasing denitrification in shallow aquatic systems.