This study evaluates the changes in the organic and nitrogen removal performance, nitrogen transformation rate, microbial enzymatic activity, oxidative stress, antioxidant response and microbial community of sequencing batch biofilm reactor (SBBR) under different ofloxacin concentrations. When the ofloxacin concentration increases from 0.2 to 20 mg/L, the organic and ammonia nitrogen removal efficiencies, nitrogen transformation rate and their corresponding enzymatic activities of SBBR gradually decrease. The presence of ofloxacin can increase the concentration of reactive oxygen species, activate antioxidant system and induce the increase of oxidative stress. These phenomena are more obvious with the increase of ofloxacin concentration in the inlet. The ofloxacin stress varies the microbial community structure, decrease the relative abundance of nitrifying genera Nitrosomonas and Nitrospira as well as denitrifying genus Denitratisoma, enrich some drug-resistant heterotrophic bacteria in SBBR, thereby weakening the ability of nitrogen removal. This study analyzes the impact of ofloxacin on the denitrification performance of SBBR from multiple perspectives, providing a theoretical basis for evaluating the ecological risks of antibiotics on wastewater biological treatment systems.