The aim of this study was to fabricate a novel bilayered wound dressing with excellent antibacterial performance through electrospinning and in situ cross-linking polymerization. Quaternary ammonium salts ([2-(methacryloyloxy)ethyl] trimethylammonium, MTA) were first polymerized and cross-linked in the presence of polycaprolactone (PCL) to form PCL/PMTA composites. Then, PCL was electrospun as the cytocompatible inner layer that directly contacts the wound, and the PCL/PMTA layer was placed as the outer layer as a defense against bacteria. The PCL/PMTA layer exhibited a hydrophilic surface and showed a strong antibacterial effect on E. coli and S. aureus. All bilayered antimicrobial nanofiber membranes did not release any toxic agents to cells, and the PCL layer exhibited better effects on cell growth. The electrospun bilayered nanofiber membranes are promising candidates for antimicrobial wound dressings.