Among the initial responses to skin injury, triggering inflammatory mediators and modifying oxidative status provide the necessary temple for the subsequent output of a new functional barrier, fibroplasia and collagen deposition, modulated by NF-κB and TGF-β1 expressions. Hence, the current study aimed to investigate the effect of local application of ursodeoxycholic acid (UDCA) on cutaneous wound healing induced in Swiss mice. Wound contraction progression was monitored by daily photographing the wounds. Enhanced fibroblast cell migration was observed after incubation with UDCA. Topical application of UDCA (500 μM) cream on excised wounds significantly enhanced wound contraction and improved morphometric scores. In addition, UDCA ameliorated the unbalanced oxidative status of granulated skin tissues. Interestingly, it showed increased expression of TGF-β1 and MMP-2 with decreased expression of NF-κB. On the other hand, UDCA significantly increased collagen fibers deposition and hydroxyproline content and enhanced re-epithelization. UDCA also modified the mitochondrial function throughout the healing process, marked by lower consumption rates of mitochondrial ATP, complex I contents as well as intracellular NAD+ contents accompanied by elevated levels of nicotinamide compared to the untreated controls. In chronic gamma-irradiated (6Gy) model, the illustrated data showed enhanced wound contraction via increased TGF-β1/MMP-2 and collagen deposition incurred by topical application of UDCA without effect on NF-κB level. In sum, the present findings suggest that UDCA may accelerate wound healing by regulating TGF-β1 and MMP-2 and fibroplasia/collagen deposition in either the two wound healing models.