Background: Open wounds leave the wound bed at risk for colonization by opportunistic pathogens. Therefore, the understanding and control of the microbial infections are of great importance for the enhanced healing and management of wounds. Reducing the wound healing time is important as it lowers the risk of infection and decreases possible complications. Grape seed extract is rich in powerful antioxidant compounds. Accordingly, grape seed extract (GSE) alone or in combination with chemical agents might be of beneficial value in wound healing. Objective: The present study aimed to evaluate the topical application of grape seed extract on wound healing of induced Staphylococcus aureus infected skin wound in diabetic rats. Methodology: Ninety male Sprague-Dawley rats weighing 150–200 grams were divided into five groups. All groups were anesthetized, shaved, and exposed to round full-thickness punch biopsy on the back: group I (control); group II (wounded); group III (wounded Staphylococcus aureus infected); group IV (wounded STZ diabetic) and group V (wounded STZ diabetic infected with Staphylococcus aureus). These last four groups were subdivided into Vaseline base treated or GSE- Vaseline treated groups. Macroscopic examination were observed at 0, 1, 3, 6, 8, 12 and 16 days after skin biopsy. Wound contraction data were expressed as a percentage of the initial wound area. Skin homogenates were prepared for determining the immunological parameters TNF-α, IL-10 TGF-β1. Results: Grape seed extract topical application improved the skin homogenate contents of TGF- β1, IL-10 and TNF-α denoting the immunological effect of GSE on wound healing in different experimental groups. Skin histopathological examination was in agreement with the immunological results. Conclusion: Grape seed extract topical application promoted skin wound contraction and closure. Furthermore, it possesses antioxidant and antibacterial properties. Grape seed extract has also strong immunoregulating properties of the skin immune system in Staphylococcus aureus infected diabetic wounded rats.