Wound healing is a dynamic, multi-phase process that includes haemostasis, tissue regeneration, cellular proliferation, and matrix modification. Traditional wound care procedures frequently encounter complications such as delayed healing and infection, demanding new therapeutic approaches. In this context, nanomaterial-based devices provide considerable benefits due to their capacity to improve medication delivery and tissue healing. We suggest a lipid-based nanofiber formulation for wound treatment that overcomes the restricted skin penetration of hydrophilic niacin, a strong wound healing agent. Niacin-loaded nanofibers (NLNFs) were manufactured utilizing glyceryl monostearate (GMS) by a self-assembly process, which included high-pressure homogenization and probe sonication for optimum nanostructure creation. The NLNFs were physicochemically characterized using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction, scanning electron microscopy (SEM) and surface profilometry to determine their morphology and homogeneity, and a drop shape analyser was used to determine hydrophobicity. In vitro tests revealed prolonged drug release, great cytocompatibility, and strong antioxidant activity, indicating superior free radical scavenging capacity. Ex vivo tests, such as the Draize skin irritation test, skin permeation test, and drug retention assays, revealed low skin irritation, increased permeability, and efficient drug retention in skin layers. In vivo experiments showed rapid wound closure and positive histological results, which were backed by hemocompatibility tests such as hemolysis and whole blood clot analysis, validating the formulation's safety. ELISA results indicated that the NLNF-treated group had higher levels of critical wound-healing indicators than the controls. Overall, our findings suggest that NLNFs have tremendous potential as a unique and effective treatment alternative for controlling and improving wound healing processes.