Novel antimicrobials with well biocompatibility are urgently needed for human public health protecting. Here, the silk fibroin (SF) nanofibrous mats coated with carboxymethyl chitosan (CMC), both extracted from natural polymers, were fabricated by combining electrospinning and electrostatic layer-by-layer (LBL) self-assembly techniques. The amphoteric CMC could be easily adsorbed on the surface of SF nanofibers due to the electrostatic interaction, which were a highly efficient and “green” route for the surface modification of SF mats. The mats after LBL procedure exhibited better hydrophilicity and stronger mechanical properties. The experimental results demonstrated that the LBL modified mats not only kept well biocompatibility but also obtained extremely enhanced antibacterial activity. More importantly, the mats displayed better bacterial inhibition with the increased CMC layers. LBL₁₀ and LBL_10.5 owned the antibacterial activity of more than 67% at the bacterial concentrations of 10⁶ cfu ml⁻¹ after 24 h cultivation, which implied that these novel natural polymer-based materials could be utilized as wound dressings for clinical skin and tissue regeneration, especially for infected wounds.