Bioretention cells (BRCs) are effective tools for treating urban stormwater, but nitrogen removal by these systems is highly variable. Improvements in nitrogen removal are hampered by a lack of data directly quantifying the abundance or activity of denitrifying microorganisms in BRCs and how they are controlled by original BRC design characteristics. We analyzed denitrifiers in twenty-three BRCs of different designs across three mid-Atlantic states (MD, VA, and NC) by quantifying two bacterial denitrification genes ( nirK and nosZ) and potential enzymatic denitrification rates within the soil medium. Overall, we found that BRC design factors, rather than local environmental variables, had the greatest effects on variation in denitrifier abundance and activity. Specifically, denitrifying populations and denitrification potential increased with organic carbon and inorganic nitrogen concentrations in the soil media and decreased in BRCs planted with grass compared to other types of vegetation. Furthermore, the top layers of BRCs consistently contained greater concentrations and activity of denitrifying bacteria than bottom layers, despite longer periods of saturation and the presence of permanently saturated zones designed to promote denitrification at lower depths. These findings suggest that there is still considerable potential for design improvements when constructing BRCs that could increase denitrification and mitigate nitrogen export to receiving waters.