Wound dressings play a crucial role in promoting wound healing by providing a protective barrier against infections and facilitating tissue regeneration. Electrospun nanofibers have emerged as promising materials for wound dressing applications due to their high surface area, porosity, and resemblance to the extracellular matrix. In this study, chitosan, a biocompatible and biodegradable polymer, was electrospun into nanofibers for potential use in wound dressing. The chitosan nanofibers were characterized by using various analytical techniques to assess their morphology and biocompatibility. Scanning electron microscopy (SEM) revealed the formation of uniform and bead-free nanofibers with diameters ranging from tens to hundreds of nanometers. Structural analysis, including Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD), elucidated the chemical composition and crystalline structure of the nanofibers. Furthermore, in vitro studies evaluated the cytocompatibility of the chitosan nanofibers with human dermal fibroblasts, demonstrating cell viability and proliferation on the nanofibers. Additionally, antibacterial properties were assessed to evaluate the potential of chitosan nanofibers in preventing wound infections. Overall, the characterization results highlight the promising attributes of electrospun chitosan nanofibers as wound dressings, paving the way for further investigation and development in the field of advanced wound care. This study has been carried out for the first time in our region and has assessed the antibacterial properties of electrospun chitosan nanofiber material. The created mat has shown efficaciousness against bacteria that are both gram-positive and gram-negative.