In this study, we investigated the potential applicability of monochloramine (MCA) for the control of membrane biofouling using a pilot-scale nanofiltration (NF) process in an advanced drinking water treatment process. It was found that membrane fouling was significantly mitigated and the increase of trans-membrane pressure was retarded more than three folds by the addition of 5 mg/L MCA. The analysis of membrane foulants after the autopsy revealed that the organic content was significantly decreased from 63.1% to 49.1% and most of the microorganisms were inactivated by the addition of MCA. Interestingly, the addition of MCA caused a dramatic decrease in protein compounds in extracellular polymeric substances extracted from biofilms, which hindered the development of dense biofilm structure. The addition of MCA also induced a significant change in the microbial community structures in the biofilm. In the presence of MCA, Alphaproteobacteria became the dominant class accounting for 59.8%, while Betaproteobacteria, which is the major bacterial community forming the biofilm in the membrane process, was decreased by 31.8%. Based on this study, the addition of MCA was effective in retarding the formation of biofilm on NF membrane surfaces by inactivating microorganisms and weakening the strength of biofilm structures.