<p>The presence of antibiotics in wastewater discharges significantly affects the environment, mainly due to the generation of bacterial populations with multiple antibiotic resistances. The cometabolic capacity of nitrifying sludge to simultaneously remove ammonium (NH₄⁺) and emerging organic contaminants (EOCs), including antibiotics, has been reported. In the present study, the removal capacity of 50&#xa0;mg ampicillin (AMP)/L by nitrifying cultures associated with biosorption and biotransformation processes was evaluated in a sequencing batch reactor (SBR) system. The contribution of nitrifying enzymes (ammonium monooxygenase (AMO) and nitrite oxidoreductase (NOR)) and β-lactamases in AMP biodegradation was evaluated using specific inhibitors in batch cultures. AMP was 100% eliminated after 5&#xa0;h since the first cycle of operation. The sludge maintained its ammonium oxidizing capacity with the total consumption of 102.0 ± 2.5&#xa0;mg NH₄⁺-N/L in 9&#xa0;h, however, the addition of AMP altered the nitrite-oxidizing process of nitrification, recovering 30 cycles later at both physiological and kinetic level. The kinetic activity of the nitrifying sludge improved along the operating cycles for both AMP removal and nitrification processes. The elimination of 24% AMP was attributed to the biosorption process and 76% to biotransformation, wherein the AMO enzyme contributed 95% to its biodegradation. Finally, the repeated exposure of the sludge to AMP for 72 operating cycles (36&#xa0;days) was not sufficient to detect β-lactamase activity. The cometabolic ability of ammonium-oxidizing bacteria for biodegrading AMP could be employed for bioremediation of wastewater.</p> Graphical Abstract <p></p>