Background: Sea buckthorn (SBT) seed oil is reported to have significant wound-healing activity. However, the oil presents with problems such as poor skin permeation and retention in the deeper layers of skin, as well as leakage, dripping, and spreading during application. These issues can be overcome by formulating nanoemulsion (NE) gel of SBT seed oil. Objective: The present study involves the development of SBT seed oil NE gels for wound healing. Materials and Methods: The NE formulations were prepared by the spontaneous emulsification method. Based on the results of pseudoternary phase diagrams, different NE formulations were prepared using SBT seed oil, surfactant/cosurfactant, and water. The selected NE formulation NEA1 was used to prepare two NE gels containing 0.5% w/w (NG1) and 1% w/w (NG2) of carbopol 940. Results: The optimized formulation NEA1 was selected on the basis of stability and conductivity studies. The NG2 NE gel was selected as the final formulation on the basis of viscosity, spreadability, extrudability, and texture analysis. This optimized NG2 formulation was further evaluated for in vivo wound-healing activity and ex vivo skin penetration studies. A significant improvement was found in NG2-treated group of animals in comparison with SBT seed oil-treated animals w.r.t wound contraction, hydroxyproline, hexosamine content, breaking strength, tensile strength, and epithelialization time of the wound. The optimized formulation also showed improved antibacterial and antifungal activity. Conclusion: The formulated NE gel of SBT seed oil showed better wound-healing, antibacterial, and antifungal activity in comparison to pure SBT seed oil.