A large number of microplastics (MPs) have been found in various stages of wastewater treatment plants, which may affect the functional microbial activity in activated sludge and lead to unstable pollutant removal performance. In this study, the effects of different concentrations of polylactic acid microplastics (PLA MPs) on system performance, nitrification and phosphorus (P) removal activities, and extracellular polymeric substances (EPS) were evaluated. The results showed that under the same influent conditions, low concentrations (50 particles/(g TS)) of PLA MPs had no significant effect on effluent quality. The average removal efficiencies of chemical oxygen demand, phosphate and ammonia were all above 80%, and the average removal efficiencies of total nitrogen remained above 70%. High concentrations (200 particles/(g TS)) of PLA MPs inhibited the activities of polyphosphate accumulating organisms (PAOs) and nitrifying bacteria. The specific anaerobic P release rate decreased from 37.7 to 23.1 mg P/(g VSS·h), and the specific aerobic P uptake rate also decreased significantly. The specific ammonia oxidation rate decreased from 0.67 to 0.34 mg N/(g VSS·h), while the change in specific nitrite oxidation rate was not significant. The dosing of PLA MPs decreased the total EPS and humic acid content. As the concentration of PLA MPs increased, microbial community diversity increased. The relative abundance of potential PAOs (i.e., Acinetobacter) increased from 0.08% to 12.57%, while the relative abundance of glycogen accumulating organisms (i.e., Competibacter and Defluviicoccus) showed no significant changes, which would lead to improved P removal performance. The relative abundance of denitrifying bacteria (i.e., Pseudomonas) decreased from 95.43% to 58.98%, potentially contributing to the decline in denitrification performance.