The performance and microbial community changes of an activated sludge sequencing batch reactor were evaluated after exposure to trimethoprim for 51 days. The average chemical oxygen demand, ammonia nitrogen, phosphorus efficiencies were 88.6% +/- 0.56%, 90.47% +/- 0.29% and 64.25% +/- 1.12%, respectively. The protein and polysaccharide contents increased with increasing trimethoprim concentration to protect the cells from the unfavorable conditions. The chemical composition of extracellular polymeric substances increased. For denitrifying bacteria, the read numbers of Pseudomonas, Flavobacterium and Bacillus were both significantly increased from Day 1 to 25 and sharply decreased by Day 50 (p < 0.05), which is consistent with the tendency of Planctomyces (Anammox). The read number of Paracoccus displayed an increasing trend, whereas Nitrospirales, Nitrospira (nitrite oxidizer) and Nitrosomonadaceae (ammonia oxidizer) were significantly decreased (p < 0.05). The read number of Rhodocyclaceae (phosphorus oxidizer) was significantly decreased from Day 1 to 25 and sharply increased by Day 50 (p < 0.05).