Ethnobotanical surveys revealed that X. orientale subsp. italicum (Moretti) Greuter has been used against central nervous system disorders in Turkish folk medicine. The aim of the present study is to verify the folkloric assertion on this plant. The compounds responsible for the activity were investigated using bioassay-guided fractionation procedures.The antidepressant activity of the aqueous, n-hexane, ethyl acetate (EtOAc), methanol (MeOH) extracts; fractions and isolated compounds from active MeOH extract were evaluated by using the in vitro MAO inhibition assay and three different in vivo models namely forced swimming test, tail suspension test, and antagonism of tetrabenazine-induced ptosis, hypothermia, and suppression of locomotor activity. The results were compared with control and reference groups, and active compounds of the plant have been determined. Through the bioassay-guided fractionation procedures, two compounds were isolated from the active fraction and their structures were elucidated by spectroscopic methods.The MeOH extract of the plant was found to possess antidepressant-like activity. This extract was then subjected to chromatographic techniques. Isolated sesquiterpene lactones were elucidated as xanthatin (1) and xanthinosin (2), which were responsible for the antidepressant-like activity.This study discovered the antidepressant potential of X. orientale subsp. italicum. Using bioassay-guided fractionation and isolation techniques, xanthatin (1) and xanthinosin (2) were determined as the main active components of the leaves.