Membrane biofouling is a critical barrier for prolonged membrane operations. In the current study, silver nanocomposite osmotic membranes were fabricated to impart anti-microbial properties. The PAN–Ag nanocomposite substrates were prepared using one-step phase inversion. With additional crosslinked layer-by-layer (xLbL) assembly on the nanocomposite substrates, xLbL3.0-Ag nanocomposite osmotic membranes were obtained. At the silver nanoparticle (AgNP) loading concentration of 0.02 wt.%, water permeability of xLbL3.0-Ag(0.02) nanocomposite osmotic membrane increased by 24.4% compared with the AgNP-free control. In addition, the silver nanocomposite membranes exhibited excellent anti-biofouling performances, with ~ 6 log colony-forming units (CFU) reduction against Gram-positive Bacillus subtilis and ~ 5 log CFU reduction against Gram-negative Escherichia coli at an AgNP loading of 0.10 wt.%. Water flux reduced to 66.9% (AgNP-free control) and 88.2% (xLbL3.0-Ag(0.10) nanocomposite osmotic membrane) of their respective original values for membranes fouled by Comamonas testosteroni I2 in PRO test mode. This study presents a convenient method to synthesize nanocomposite osmotic membranes with excellent anti-biofouling property for potential use for pressure retarded osmosis (PRO). Our results provide significant implications for anti-fouling nanocomposite osmotic membrane fabrication as well as PRO fouling control.