Background:Easy availability, relatively low cost with fewer side effects, has made the herbal extracts/fractions/pure compounds as prominent source of medicinally important molecules. Dioscorea villosa L. commonly known as wild yam belongs to the family Dioscoreaceae and has been used in various parts of India to treat joint pain, arthritis, and various other diseases. However, its role in wound healing has not been documented so far. In the current study, the in vitro wound healing capabilities of D. villosa were examined using L929 cells. Materials and Methods:Methanolic extraction of D. villosa leaves was prepared by applying inexpensive maceration method. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to analyze the cytotoxicity of D. villosa extract and in vitro wound healing capabilities were investigated by applying scratch assay. The qualitative measurement of different secondary metabolites was determined by standard biochemical assays. Gas chromatography-mass spectrometry (GC-MS) was performed to identify the possible wound healing components present in the methanolic leaf extract of D. villosa, and the antioxidant properties of the plant extract were evaluated by α,α-diphenyl-β-picrylhydrazyl and ferric reducing antioxidant power assays. Furthermore, the possible molecular factors involved in the proliferation and migration of fibroblast in the presence of D. villosa extract was determined by flow cytometry technique. Results:The experiments to analyze the cytotoxic effect of D. villosa on L929 cells revealed that at the highest concentration used, i.e., 500 μg/mL after 48 h of incubation, 96.06% ± 0.42% of the cells were viable. The results of the scratch assay revealed that 125 μg/mL of plant extract induced the migration in 88.58% of fibroblast cells. Through GC-MS analysis, antioxidant and anti-inflammatory molecules such as 1 H-Indole-2,3-dione (Isatin) and Dexamethasone have been identified. In addition, flow cytometry data showed the influence of plant extract on the expression of Collagen-1 and transforming growth factor (TGF)-beta, which play a major role in the wound healing processes. 125 μg/mL of plant extract induced Collagen-1 in 22.18% cells and TGF-beta in 80.77% of cells, respectively. Conclusion:The presence of potent antioxidant and anti-inflammatory molecules and capability to induce the expression of fundamental wound healing molecular factors TGF-beta and collagen-1 in fibroblast cells, endorsed D. villosa as a potential wound healing agent.