Elevated atmospheric carbon dioxide concentrations ([CO2]) might change the abundance and the function of soil microorganisms in the depth profile of agricultural soils by plant-mediated reactions. The seasonal pattern of abundance and activity of nitrate-reducing bacteria was studied in a Mini-FACE experiment planted with oilseed rape (Brassica napus). Three depths (0–10, 10–20 and 20–30 cm) were sampled. Analyses of the abundances of total (16S rRNA gene) and nitrate-reducing bacteria (narG, napA) revealed strong influences of sampling date and depth, but no [CO2] effects. Abundance and activity of nitrate reducers were higher in the top soil layer and decreased with depth but were not related to extractable amounts of nitrogen and carbon in soil. Dry periods reduced abundances of total and nitrate-reducing bacteria, whereas the potential activity of the nitrate reductase enzyme was not affected. Enzyme activity was only weakly correlated to the abundance of nitrate-reducing bacteria but was related to NH4+ and NO3− concentrations. Our results suggest that in contrast to the observed pronounced seasonal changes, the elevation of atmospheric [CO2] has only a marginal impact on nitrate reducers in the investigated arable ecosystem.