Woodchip denitrification bioreactors, a relatively new technology for edge-of-field treatment of subsurface agricultural drainage water, have shown potential for nitrate removal. However, few studies have evaluated the performance of these reactors under varied controlled conditions including initial woodchip age and a range of hydraulic retention times (HRTs) and temperatures similar to the field. This study investigated (i) the release of total organic C (TOC) during reactor start up for fresh and weathered woodchips, (ii) nitrate (NO3–N) removal at HRTs ranging from 2 to 24 h, (iii) nitrate removal at influent NO3–N concentrations of 10, 30, and 50 mg L−1, and (iv) NO3–N removal at 10, 15, and 20°C. Greater TOC was released during bioreactor operation with fresh woodchips, whereas organic C release was low when the columns were packed with naturally weathered woodchips. Nitrate-N concentration reductions increased from 8 to 55% as HRT increased. Nitrate removal on a mass basis (g NO3–N m−3 d−1) did not follow the same trend, with relatively consistent mass removal measured as HRT increased from 1.7 to 21.2 h. Comparison of mean NO3–N load reduction for various influent NO3–N concentrations showed lower reduction at an influent concentration of 10 mg L−1 and higher NO3–N reductions at influent concentrations of 30 and 50 mg L−1. Nitrate-N removal showed a stepped increase with temperature. Temperature coefficient (Q10) factors calculated from NO3–N removal rates ranged from 2.2 to 2.9.