•Role of thymoquinone treated with cold plasma (TQcp) on the rat wound model.•TQcp significantly reduced the skin content of tumor necrosis factor- α.•TQcp could up-regulate the skin levels of transforming growth factor-beta.•TQcp significantly enhanced the skin content of interleukin-10, alpha smooth muscle actin and vascular endothelial growth factor.•TQcp could improve the histopathological and ultra-structural picture of the skin.•TQcp revealed a significant potential for wound healing. Wound healing is a series of coordinated events that involve tissue repair and regeneration. Cold atmospheric plasma approach sheds the light on the mechanism that initiates the inflammatory responses throughout the healing cascade. The present study was planned to assess the effect of thymoquinone treated with cold plasma (TQcp) on the rat wound model compared to thymoquinone (TQ). To assess the wound healing potential of TQcp, a full-thickness wound model was used. The induced wound was smeared, starting just after excision, twice daily with TQcp and TQ for 7 days. Our findings revealed that TQcp improved the skin healing potential by augmenting the skin regeneration indices as evidenced by enhancing the new production of hyaluronic acid and collagen type I. TQcp significantly reduced the skin content of tumor necrosis factor- α and inhibited the hypertrophic scarring by up-regulating the skin content of transforming growth factor-beta. Furthermore, TQcp enhanced the levels of interleukin-10, alpha smooth muscle actin and vascular endothelial growth factor, demonstrating a great potential for wound healing that also reflected in the histopathological and ultra-structural picture of the skin. Finally, our results demonstrated that TQcp revealed a significant potential for wound healing than TQ alone.