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The cometabolism of ammonia‐oxidizing bacteria (AOB) in nitrifying activated sludge can effectively degrade antibiotics. However, the roles of other microorganisms in antibiotic removal processes remain uncharacterized. In this study, the effects of AOB, nitrite oxidizing bacteria (NOB), and heterotrophs on the removal of the antibiotic trimethoprim (TMP) at environmentally relevant concentrations in an enriched nitrifying activated sludge are quantified. AOB is found to be the most efficient for TMP removal, which is related with ammonium concentration. TMP removal is increased from 48.0% to 64.0% as the ammonium concentration increased from 50 to 150 mg L−1. However, with a further increase in ammonium concentration to 250 mg L−1, TMP removal is decreased to 45.6%. The percentages of TMP removed by AOB, NOB, and heterotrophs are quantified by batch experiments, and are 78.7%, 18.6%, and 2.7%, respectively. The results demonstrate that in nitrifying activated sludge system, the contribution of NOB and heterotrophs on TMP removal should also be considered.