This study aims to evaluate the Vancomycin (VCM) combination with Chitosan (CS)/ Polyethylene oxide (PEO) nanofibers’ intrinsic antibacterial properties causing a synergistic effect against possible serious bacterial infections (PSBI). VCM/CS nanofiber scaffold was fabricated using the electrospinning method. Characterizations are performed by Fourier transform infrared (FT-IR) to examine the functional groups of each compound, scanning electron microscopy (SEM), and transient electron microscopy (TEM) to evaluate nanofiber diameter and structure. Antibacterial activities of the nanofibrous scaffold were assessed against bacterial strains, including standard Staphylococcus aureus ( S. aureus), VCM-sensitive Enterococcus (VSE), methicillin-resistant S. aureus (MRSA), VCM-resistant Enterococcus (VRE), and Streptococcus group A by microdilution broth methods. The FT-IR, SEM, and TEM examination results confirm the CS/PEO nanofiber scaffold fabrication. The antibacterial examination results showed no significant difference between the minimum inhibitory concentration (MIC) values of VCM and with MIC of VCM/CS nanofibers. Still, there were significant differences between the MIC of CS and VCM/CS nanofibers in S. aureus, but this is not more significant than VCM. This study illustrated that VCM coupled to CS nanofibers had acceptable antibacterial activity against the Gram-positive bacterium. This work motivated researchers’ insight into nanostructures’ potential accompanied by antibacterial polymer and antibiotics synergistic effects against PSBI.