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The phytochemical analysis of the investigated Immortelle essential oil revealed the presence of monoterpenes and sesquiterpenes as major components that might be efficient as a wound healing potential agent. The present study aimed to develop an ointment based on the Immortelle essential oil and investigate its wound healing effects on excision wounds in diabetic rats. The topical formulated Immortelle ointment was subjected to pharmaco-technical characterization. Thirty-two diabetic rats with the induced excision wound were used to evaluate in vivo wound healing effects of ointment. The animals were randomly divided into four groups untreated or topically treated with either a 1% silver sulfadiazine, the ointment base, or Immortelle ointment. The response to the treatment was assessed by macroscopic, biochemical and histopathological analysis. The ointment, compatible with the skin remained stable for 6 months. Topical application of the Immortelle ointment showed the highest wound contraction with the highest content of hydroxyproline in comparison to the all examined groups. The Immortelle ointment showed significant wound contraction from day 7 to day 21 as compared to other groups. On the day 21, there was an average of 99.32% wound contraction in the Immortelle group, whereas the mean wound contraction in the negative control and ointment base group was 71.36% and 81.26% respectively. The histopathological results validated the potential wound healing effect of Immortelle ointment with evident post-excision scar maturation and increased collagen fibers density. Our findings revealed that the Immortelle ointment approach might serve as a promising and innovative tool for wound healing.