Enhancing antimicrobial activity is an effective strategy to mitigate membrane biofouling. In this study, an antimicrobial electrospun polyacrylonitrile (PAN) nanofiber mat decorated with in situ synthesized silver nanoparticles (AgNPs) was developed as support for a thin-film composite forward osmosis (FO) membrane. Effects of AgNPs on the physicochemical properties, water flux, reverse salt flux, and antimicrobial activity of the FO membrane were investigated, and also the antimicrobial mechanism was explored. Results showed AgNPs were monodisperse in the nanofibers, and the Ag/PAN nanofiber support (Ag/PAN-NFS) had a scaffold-like structure which could reduce internal concentration polarization. The Ag/PAN-NFS supported thin-film nanocomposite (Ag/PAN-TFN) FO membrane demonstrated outstanding hydrophilicity, resulting in high water fluxes in both FO and PRO modes. The Ag/PAN-TFN FO membranes, even the one with the lowest silver content of 0.5 wt %, exhibited excellent antibacterial activities for E. coli (96%) and S. aureus (92%), which was mainly due to the Ag⁺-species released into the solution.