Reverse osmosis (RO) membranes with high separation performance and fouling resistance are essential for practical water treatment applications. In this study, we introduce a biocidal surfactant, to the interfacial polymerization process to fabricate high-performance and fouling-resistant RO membranes. The surfactant reduces the interfacial tension at the water–organic interface, thereby enhancing the diffusion of amine monomers to the organic phase, which leads to the formation of a highly dense and permeable polyamide (PA) selective layer. Simultaneously, surfactant embedded within the PA matrix imparts hydrophilic and antibacterial properties to the membrane, enhancing the organic/biofouling resistance of the resultant membrane. This strategy provides a facile and sustainable route for tailoring the performance and functionality of membranes for diverse separation applications.