Wood chip bioreactors are receiving increasing attention as a means of reducing nitrate in subsurface tile drainage systems. Agrochemicals in tile drainage water entering wood chip bioreactors can be retained or degraded and may affect denitrification. The degradation of 5 mg L −1 atrazine, enrofloxacin, and sulfamethazine under denitrifying conditions in wood chips from an in situ reactor was determined. The impact of these chemicals on denitrifying microorganisms was assessed using the denitrification potential assay, most probable number (MPN), and quantitative polymerase chain reaction targeting the nosZ gene of the denitrifiers. Initial half‐lives for these chemicals in the aqueous phase were 0.98 d for atrazine, 0.17 d for enrofloxacin, and 6.2 d for sulfamethazine. Similar rates of disappearance in autoclaved and nonautoclaved wood chip solutions during the first 48 h suggested sorption was the dominant mechanism. The presence of atrazine did not impair denitrification potential, the MPN, or the nosZ copy number. The denitrifier MPN and nosZ copy number in sulfamethazine‐ and enrofloxacin‐treated microcosms were less than the control within the first 5 d after chemical addition, whereas the denitrification potentials were not affected. However, after 45 d the denitrification rate, MPN and nosZ gene copy numbers for sulfamethazine and enrofloxacin were similar to that of the no‐chemical control, indicating that acclimation of the denitrifier population to the antibiotic or reduced bioavailability over time allowed recovery of the denitrifier population.