Biosafe antibacterial agents are urgently demanded in treating infection especially chronic infection. However, efficient and controlled release of those agents remains great challenging. Two nature-derived agents, lysozyme (LY) and chitosan (CS), are selected to establish a facile method for long-term bacterial inhibition. We incorporated LY into the nanofibrous mats, then deposited CS and polydopamine (PDA) on the surface by layer-by-layer (LBL) self-assembly. In this vein, LY is gradually released with the degradation of nanofibers, and CS is rapidly disassociated from the nanofibrous mats to synergistically result in a potent inhibition against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) over a period of 14 days. Besides long-term antibacterial capacity, LBL-structured mats could readily achieve a strong tensile stress of 6.7 MPa with an increase percentage of up to 103%. The enhanced proliferation of L929 cells arrives at 94% with help of CS and PDA on the surface of nanofibers. In this vein, our nanofiber has a variety of advantages including biocompatibility, strong long-term antibacterial effect, and skin adaptability, revealing the significant potential to be used as highly safe biomaterial for wound dressings.