Denitrification bioreactors to reduce the amount of nitrate-nitrogen in agricultural drainage are now being deployed across the U.S. Midwest. However, there are still many unknowns regarding internal hydraulic-driven processes in these engineered treatment systems. To improve this understanding, the internal flow dynamics and several environmental parameters of a denitrification bioreactor treating agricultural drainage in Northeastern Iowa, USA were investigated with two tracer tests and a network of bioreactor wells. The bioreactor had a trapezoidal cross section and received drainage from approximately 14.2 ha at the North East Research Farm near Nashua, Iowa. It was clear from the water surface elevations and the continuous pressure transducer data that flow was attenuated within the bioreactor (i.e., reduction in peak flow as the hydrograph moved down gradient). Over the sampling period from 17 May to 24 August 2011, flow conditions and internal parameters (temperature, dissolved oxygen, oxidation reduction potential) varied widely resulting in early samplings that showed little nitrate removal ranging to complete nitrate removal (7–100% mass reduction; 0.38–1.06 g N removed per m³ bioreactor per day) and sulfate reduction at the final sampling event. The bioreactor's non-ideal flow regime due to ineffective volume utilization was a major detriment to nitrate removal at higher flow rates. Regression analysis between mass nitrogen reduction and theoretical retention time (7.5–79 h) suggested minimum design retention times should be increased, though caution was also issued about this as increased design retention times and corresponding larger bioreactors may exacerbate detrimental by-products under low flow conditions. Operationally, outlet structure level management could also be utilized to improve performance and minimize detrimental by-products.