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Burns are one of the most devastating traumatic injuries, and in cases of advanced burns, patients require urgent and specialized care to minimize mortality. Studies have shown that a variety of herbal medicines and nanoparticles can be used to treat burn wounds. Among them mixture of Eisenia fetida oil, valerian, and zinc oxide (ZnO) nanoparticles can be a good choice due to their healing properties. This study aimed to synthesize an ointment based on Eisenia fetida oil, Valeriana officinalis, and zinc oxide to investigate its effect on skin wound healing. The hemocompatibility and biocompatibility of the designed ointment were evaluated using blood clotting index (BCI), hemolysis test, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. The antibacterial effects of the designed ointment were evaluated using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill tests. Finally, the healing ability of the designed ointment was evaluated in a rat model by creating a 3 × 3 cm burn wound. Wound healing was evaluated after 14 days by hematoxylin-eosin (H&E) and Verhoeff-Van Gieson (VVG) staining. The results showed that the addition of Valeriana officinalis and zinc oxide to Eisenia fetida oil improved hemocompatibility, biocompatibility, improved cell migration, and improved antibacterial properties. Additionally, the results of H&E and VVG staining demonstrated the good efficacy of the designed ointment in increasing epidermal thickness, promoting angiogenesis, and enhancing collagen and elastin fibers. These results indicate the potential of the designed ointment as a promising tool for skin wound healing and clinical trials.