The genus Prunella L. (Lamiaceae) is represented by nine species in the world and four species in Turkey. The infusion prepared from the aerial parts of Prunella vulgaris L. is used internally for abdominal pain and as an expectorant, the decoction prepared from all parts is used internally or externally as a wound healing. This study aims to investigate the wound healing potential of Prunella vulgaris L. on the scientific platform. The aerial parts of the plant were extracted with 80% methanol. The resulting aqueous methanol extract was partitioned with n-hexane and ethyl acetate, and sub-extracts were obtained. The wound healing effects of the methanol extract and sub-extracts were studied in mice and rats using linear incision and circular excision wound models, and the anti-inflammatory effect was investigated using acetic acid-induced capillary permeability test. Isolation studies were performed using the ethyl acetate sub-extract, which exhibited the highest activity. Using various chromatographic methods, 6 compounds were isolated from the ethyl acetate sub-extract. The structures of the compounds were identified as methyl arginolate, ursolic acid, chlorogenic acid, rosmarinic acid, methyl 3-epimaclinate, and ethyl rosmarinate by spectroscopic techniques (UV, IR, 13C-NMR, 1H-NMR, 2D-NMR, MS). The wound healing mechanisms of the pure compounds were investigated by performing assays to inhibit the enzymes hyaluronidase, collagenase, and elastase. Ursolic acid, chlorogenic acid, and rosmarinic acid were found to be responsible for the anti-inflammatory and wound healing effects. The experimental study revealed that Prunella vulgaris showed significant wound healing and anti-inflammatory activities.