Nanofiltration (NF) shows great potential for water treatment and recycling. Nevertheless, severe membrane fouling significantly decreased lifetime and performance of NF membranes, impeding NF applications for clean water production. Herein, novel antifouling NF membranes with a pseudo zwitterionic separation layer were designed using layer-by-layer (LBL) assembly of amino-quinone networks (AQNs) and hydrophilic phytic acid (PhA) (AQN-PhA). The AQNs were formed by 5-hydroxy-1,4-naphthoquinone (HNQ) and polyethylenimine (PEI) via Michael addition and Schiff base reaction. The modification conditions of the AQN-PhA system were systematically investigated for an optimum membrane performance. The optimized conditions were found to be a PEI molecular weight of 600 Da, HNQ/PEI ratio at 1:2, an AQN coating time of 10 min, and pH of PhA at 5. The optimized membrane (i.e., AQN-PhA-5-2L) showed improved hydrophilicity along with a pure water permeability of 9.0 L•m −2 •h −1 •bar −1 and a molecular weight cut-off of 766 Da. It exhibited excellent fouling resistance with approximately 94% flux recovery ratio against a synthetic wastewater during a long-term filtration, which was superior in comparison with a benchmark commercial polyamide NF membrane. This study provides an idea for simple, feasible, and scalable zwitterionic modification in the future design of antifouling NF membranes.