Silk fibroin (SF) is increasingly needed in tissue engineering for its superior biocompatibility. However, the practical applications of pure SF biomaterials confront bacterial infection problems. In this study, chitosan (CS) and polydopamine (PDA) were introduced into electrospun nanofibrous SF mats through layer-by-layer self-assembly (LBL) to obtain enhanced antibacterial ability and cytocompatibility. The surface morphology and composition analysis confirmed the successful deposition. After depositing 15 bilayers, the tensile modulus of the mats in wet condition increased from 2.16 MPa (pristine SF mats) to 4.89 MPa. A trend towards better hydrophilicity performance was also recorded with more bilayers coating on the mats. Besides, LBL structured mats showed improved antibacterial ability of more than 98 % against E. coli and S. aureus. In addition, advancement in biocompatibility was observed during the proliferation experiment of L929 cells. Overall, the deposition of CS and PDA may further expand the use of SF in biomedical field.