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Nanofiltration membranes have evolved as a promising solution to tackle the clean water scarcity and wastewater treatment processes with their low energy requirement and environment friendly operating conditions. Thin film composite nanofiltration membranes with high permeability, and excellent antifouling and antibacterial properties are important component for wastewater treatment and clean drinking water production units. In the scope of this study, thin film composite nanofiltration membranes were fabricated using polyacrylonitrile (PAN) support and fast second interfacial polymerization modification methods by grafting polyethylene amine and zwitterionic sulfobutane methacrylate moieties. Chemical and physical alteration in structure of the membranes were characterized using methods like ATR-FTIR spectroscopy, XPS analysis, FESEM and AFM imaging. The effects of second interfacial polymerization to incorporate polyamide layer and ‘ion pair’ characteristics, in terms of water contact angle and surface charge analysis was investigated in correlation with nanofiltration performance. Furthermore, the membrane characteristics in terms of antifouling properties were evaluated using model protein foulants like bovine serum albumin and lysozyme. Antibacterial properties of the modified membranes were investigated using E. coli as model biofoulant. Overall, the effect of second interfacial polymerization without affecting the selectivity layer of nanofiltration membrane for their potential large-scale application was investigated in detail.