In shallow-water systems with calm hydrodynamic, dense vegetation stands provide most of the available surface for periphyton development. The large ratio between biological active surfaces and water volume amplifies the influence of biofilm activity on water chemistry, resulting the key factor responsible for nitrogen removal performance of wetlands and waterways. However, the denitrification capacity of biofilms on emergent macrophytes remains understudied, especially if investigated on dead stems during the non-vegetative season. The aims of the present study were: 1) to quantify the role of biofilms colonizing dead stems of Phragmites australis in NO3−mitigation via denitrification in winter (∼11 °C) in a NO3−-rich drainage canal; 2) to determine how the biofilm denitrifying capacity varies as a function of water velocity (0–6 cm s−1). Denitrification was assessed by the concomitant measurements of NO3−consumption and N2production from analyses of N2:Ar by Membrane Inlet Mass Spectrometry. Sediments with biofilms were found more efficient in converting NO3−to N2(7–17 mmol N m−2d−1) than bare sediments (3–5 mmol N m−2d−1). Denitrification activity in biofilms responded positively to increasing water velocity that enhanced the rate of NO3−supply to the active surfaces. Results of the present study showed that denitrification performed by biofilms on senescent stems proceeds beyond the vegetative season throughout the cold period and maintains the depuration capacity when drainage canals may still drive high NO3−loads leached from the agricultural fields. The development of a diversified and extended microbial community throughout the year together with water velocity should be taken into account as key elements in the management of the canal networks aimed at combining hydrological needs and water quality goals.