Amoxicillin (AMX) is one of the most widely used antibiotics in the world and its presence in wastewater is of great concern for its potential to bacteria selection. However, there is still a gap about the toxicity effect of AMX in nitrifier biomass from activated sludge (AS). This study is based on the implementation of respirometric tests in batches in order to evaluate the toxic effluent toxicity in the nitrification process of AS. The tests were conducted by comparing respiration rates with effluent containing ammonia nitrogen (NH 4 + -N) and nitrite nitrogen (NO 2 − -N) called "reference" and batches containing toxic effluent doped with different concentrations of AMX here called "process." Results with effluent containing concentrations greater than 100 mg L −1 showed that AMX negatively affected the specific growth rate (μ m ) of ammonia-oxidizing bacteria (AOB) (from 0.50 d −1 to 0.13 d −1 ) and nitrite-oxidizing bacteria (NOB) (from 0.64 d −1 to 0.15 d −1 ). Although there is no total inhibition of populations, these μ m values are limiting for a feasible development of the nitrification process in AS systems. The removal of AMX decreased from 99 to 37% (liquid phase) when the concentration of AMX increased (20 mg L −1 to 200 mg L −1 ). A decrease in the microbial community AOB and NOB was observed through fluorescent in situ hybridization (FISH), corroborating the results of respirometry. In summary, the study showed that the inhibition of the AS nitrification process occurs in the presence of high concentrations of AMX and the most susceptible group are the NOB.