Introduction. Opuntia ficus indica L. inermis (OFI) is used in traditional medicine pharmacopeia for its richness in natural bioactive compounds. It has been proven to be effective in the improvement of the healing of laser-induced skin burns. The aim of the present study was to investigate the wound healing effect of OFI extracted oil on full-thickness skin wound. Materials and Methods. The OFI seeds were firstly isolated from mature prickly pears, washed, dried, and then cold-pressed. The antimicrobial activities of OFI seed oil were estimated in vitro against bacteria, yeast, and fungi. Minimum Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) were calculated. Skin wound healing was investigated using an excisional wound healing model in rats. The skin wounds of three experimental groups of rats were topically treated once/day with saline solution (control group), 0.15 mg/mm2 of a reference drug Esth’Elle Pharma Cicaplaie cream (reference group), and 0.6 μl/mm2 of OFI seed oil (OFI oil group). The healing process was monitored daily and the percentage of wound contraction was calculated. A histological study was carried on skin biopsies. Results. The extracted oil has shown an interesting antimicrobial effect on Enterobacter cloacae, antiyeast effect against Candida parapsilosis and Candida sake, and antifungal activity against three opportunistic cutaneous molds (Penicillium, Aspergillus, and Fusarium). Moreover, OFI oil has shown a good wound healing effect. It prevents cutaneous infections and reduces the reepithelialization phase. Conclusion. OFI extracted oil has in vitro antimicrobial/fungal properties and in vivo wound healing activity. It seems to be efficient in the treatment of cutaneous infections and the promoting of the scarring process.