The wide utilization of zinc oxide nanoparticles (ZnO NPs) in many products could cause their release into wastewater treatment plants. Although the potential effects of ZnO NPs on synthetic wastewater treatment have been studied, reports concerning their impacts on biological nitrogen (N) and phosphorus (P) removal from real wastewater are sparse, and the related mechanisms of these effects are still unclear. In this study, the influences of ZnO NPs on nitrogen and phosphorus removal from real municipal wastewater were investigated, and the results were compared with those using synthetic wastewater. The data showed that 50 mg/L ZnO NPs did not affect the settleability and membrane integrity of activated sludge fed with real or synthetic wastewater. Although the deterioration of N and P removal from both wastewaters was observed, the ZnO NPs-induced inhibition to real wastewater treatment was smaller than that to synthetic wastewater treatment (36% versus 100% inhibition to phosphorus removal). Fluorescence excitation-emission matrix (EEM) analysis showed that the real wastewater contained higher dissolved organic matters such as humic substances than the synthetic one. The presence of dissolved organic matter decreased the dissolution of ZnO NPs, which could lower the ZnO NPs-induced negative effects on biological N and P removal from real wastewater. Further experiments revealed that ZnO center dot NPs showed smaller inhibition to the catalytic activities of nitrate reductase, exopolyphosphatase, and polyphosphate kinase of activated sludge cultured by real wastewater, which was another reason for the lower influence of ZnO NPs on biological N and P removal from real wastewater.