Anti-biofouling nanofiltration (NF) membranes can reduce the damage of bacteria to membranes and are typically fabricated by grafting bactericidal chemicals on membrane surface, with potential environmental safety issues as well as health hazards. Vanillin (VA) is a natural non-toxic bacterial inhibitor that has been proven to inhibit a wide range of bacteria. In this study, VA was introduced as an aqueous phase additive to the selective layer by interfacial polymerization reaction thus preparing NF membranes with excellent anti-biofouling properties. The biofilm area on the optimized membrane surface formed by Bacillus subtilis and Escherichia coli were reduced by 66.0 % and 75.1 %, respectively. In addition, the hydrophilicity enhancement and the membrane surface roughness reduction were attributed to the VA introduction, which resulted in an improvement of the anti-adhesion properties of modified membranes, and flux recovery ratio of optimal membranes was 75.7 % while flux decline ratio was 50.4 %. The water permeability for mixed salt solution of membrane with the optimized VA concentration was 1.22 times higher than control membrane and rejection of NaSO4 was 99.0 %, meanwhile the separation factor of NaCl/Na2SO4 stabilized in the range of 27.4–28.0. This facile process provided an non-biotoxic method to prepare environmentally friendly and effective anti-biofouling membranes.