Ethnobotanical surveys revealed that Daphne oleoides Schreber has been used against rheumatic pain and for wound healing in Turkish folk medicine. The aim of the present study is to verify the folkloric assertion of D. oleoides ssp. oleoides (DOO) by bioassay-guided fractionation procedures leading to determination of the active component(s) and to elucidate the activity mechanisms of the isolated compounds. The wound healing activity of the methanol extract, its subextracts, fractions and isolates was evaluated by using two different in vivo wound healing experimental techniques. Anti-inflammatory and antioxidant activities of the test materials were also evaluated. For the determination of the activity mechanisms, the isolated compounds were screened for hyaluronidase, collagenase and elastase enzyme inhibitory activities. The methanolic extract of DOO was found to possess potent wound healing activity. This extract was then subjected to successive solvent extractions with n-hexane, dichloromethane, ethyl acetate (EtOAc) and n-butanol. EtOAc subextract yielded three compounds, quercetin 3-O-glucoside, triumbellin and rutarensin by using chromatographic separation techniques. The experimental study revealed that D. oleoides subsp. oleoides methanolic extract possesses significant wound healing effect and quercetin 3-O-glucoside was determined as the active component responsible from the activity.