Objective: To evaluate the in vivo wound healing activity of Acanthus leucostachyus leaf extracts using an excision wound model in mice. Methods: Mice were divided into two groups of six animals in each group: the control group and the Acanthus leucostachyus extract-treated group. Healing potential was evaluated by determination of physical parameters (contraction rate, epithelialization period, and tensile strength), biochemical parameters (protein, DNA, and hydroxyproline content), the expression of growth factor and pro-inflammatory cytokines, as well as histological and ultrastructural observations. Results: Treatment with Acanthus leucostachyus leaf extracts markedly increased the rate of wound contraction, tensile strength, the concentrations of protein, DNA, and hydroxyproline, and the expression of growth factor, as well as promoted epithelialization, compared to the control. In addition, Acanthus leucostachyus leaf extracts significantly reduced the expression of pro-inflammatory cytokines. Histological and ultrastructural studies revealed the presence of thicker epithelial layer and smoother surface topography in the extract-treated group compared to the control. Conclusions: Acanthus leucostachyus leaf extracts show potent wound-healing activity and can be used as a wound healing agent.