Antibiotics released in agricultural soils alter soil bacterial communities, inducing antimicrobial resistance and, in turn, canceling the efficiency of antibiotic drugs used for human and animal health. In soils, antibiotic impact on nitrogen cycling is poorly known, notably when antibiotic mixtures are applied. We hypothesized that the impact of antibiotic mixtures would have higher effects on denitrification. We exposed soil denitrifying bacteria enrichments to tetracycline, ofloxacin, sulfamethoxazole and tylosin, either applied single or as mixture of three antibiotics, during 7 days under denitrifying conditions. We measured the minimum inhibitory concentration of the N2O-reducing capacity of the bacterial enrichment, we deduced the half maximal effective concentration (EC50) from the experimental data and from the concentration addition hypothesis, and we quantified nosZ gene abundances. Results show that single antibiotic exposure inhibited N2O-reduction only for tetracycline at 64 mg/L. Inhibition by antibiotic mixtures always exceeded the modeled inhibition calculated by concentration addition. At high-antibiotic exposure, nosZ gene clade I denitrifiers remained abundant, of 107–108 copies/ng DNA. NosZ gene clade II denitrifiers increased with antibiotic concentrations. Our findings reveal for the first time the synergistic effects of antibiotic mixtures on soil nitrogen cycling.