AbstractGelatin (GE) nanofiber suspensions prepared by the extrusion of an immiscible polymer solution blend were cast into nanofibrous membranes. An organic chemical capable of generating radicals under photoexposure, anthraquinone‐2,6‐disulfonic acid (AQS), was assembled layer by layer (LBL) onto the gelatin nanofiber membranes. The composite gelatin nanofibrous membranes assembled with AQS demonstrated excellent photoinduced antibacterial properties by killing bacteria. Scanning electron microscopy images demonstrated that the composite nanofibrous membranes maintained a well‐defined nanofiber morphology and large specific surface area after LBL assembly. Moreover, the nanofiber morphology and nanofibrous structure of the composite nanofibrous membranes were completely reserved after photoexposure. An analysis of the cell indicated that the composite gelatin nanofibrous membranes assembled with AQS exhibited the same biocompatibility as the original gelatin nanofibers. These results demonstrate that AQS LBL‐assembled gelatin nanofibrous membranes could be a potential candidate for wound dressing applications. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013