Polyamide (PA) nanofilm membranes are key materials for water treatment and desalination. Although surfactants are widely used to tune the interfacial polymerization (IP) process for PA nanofilm formation, their regulatory mechanisms remain incompletely understood. Herein, we synthesized PA nanofilm membranes at a support-free interface in the presence of anionic surfactants with different tail lengths and counterions and comprehensively characterized them to identify the IP mechanism regulated by the surfactant structure. The use of the surfactant with a longer alkyl tail or a smaller/weaker-bound counterion resulted in greater alterations in the structural and physicochemical properties and separation performance of the PA nanofilm membranes. Collaborated with computational simulations, this phenomenon could be attributed to solutal Marangoni instability triggered by the interfacial migration (via binding to PA) of the surfactant complexed with amine monomers during IP. A longer alkyl chain or smaller/weaker-bound counterion of the surfactant intensified Marangoni instability by promoting the interfacial formation of surfactant–amine complexes and/or facilitating their binding to PA, leading to greater PA deformations. Our work provides new insights into the surfactant-regulated IP mechanism as well as a facile means to rationally tailor the properties and performance of IP-assembled nanofilm membranes for various environmental applications.

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