Traditional Medicine is widely used in clinical research due to its low toxicity, low number of side effects, and low cost. Many components of common fruits and vegetables play well-documented roles as chemo preventive or chemotherapeutic agents that suppress tumor genesis. The present study was aimed to identify the DPPH (2, 2-diphenyl-1-picrylhydrazyl) free-radical scavenging constituents from methanol extract of Launaea mucronata using bioassay-guided fractionation. The methanolic extract revealed a strong antioxidant activity, the IC50 values (the concentration with scavenging activity of 50%) was 25.94 mg/ml. The fractionation of the active methanol extract by silica gel column chromatography revealed compounds with strong antioxidant activity. The isolated bioactive compound was determined as, apigenin, quercetin, rutin, isorhamnetin-3-O-β-glucoside, quercetin-3-O-β-glucoside, apigenin-7-O- β-diglucoside, kaempferol-3-o-rutinoside), by comparing spectral data (UV, IR, 1H NMR, 13C NMR, and MS) with literature reports. The results obtained confirmed that methanol extract of Launaea mucronata or its derived phytocompounds can be used potentially as a bioactive source of natural antioxidants by contributing beneficial health effects. Launaea mucronata was in vitro investigated for cytotoxic activity against HCT116, HepG2, Hella and MCF-7 cell lines, doxorubicin was used as the reference drug. The results show IC50 19, 19.60, 12.60 and 12.70 μg/ml respectively.