Wound infections are still problematic in many cases and demand new alternatives for current treatment strategies. In recent years, biomaterials-based wound dressings have received much attention due to their potentials and many studies have been performed based on them. Accordingly, in this study, we fabricated and optimized an antibacterial chitosan/silk fibroin (CS/SF) electrospun nanofiber bilayer containing different concentrations of a cationic antimicrobial peptide (AMP) for wound dressing applications. The fabricated CS/SF nanofiber was fully characterized and compared to the electrospun silk fibroin and electrospun chitosan alone in vitro. Then, the release rate of different concentrations of peptide (16, 32, and 64 µg/ml) from peptide-loaded CS/SF nanofiber was investigated. Finally, based on cytotoxic activity, the antibacterial activity of scaffolds containing 16 and 32 µg/ml of the peptide was evaluated against standard and multi-drug resistant strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa isolated from burn patients. The peptide-loaded CS/SF nanofiber displayed appropriate mechanical properties, high water uptake, suitable biodegradation rate, a controlled release without cytotoxicity on Hu02 human foreskin fibroblast cells at the 16 and 32 µg/ml concentrations of peptide. The optimized CS/SF containing 32 μg/ml peptide showed strong antibacterial activity against all experimental strains from standard to resistance. The results showed that the fabricated antimicrobial nanofiber has the potential to be applied as a wound dressing for infected wound healing, although further studies are needed in vivo.