The aim of this study was to isolate and identify secondary metabolites from Sonchus brachyotus and evaluate their antioxidant and anti-inflammatory activities. In this study, we isolated three flavonoids from a 70% EtOH extract by Medium Pressure Liquid Chromatography (MPLC) and prep-High-Performance Liquid Chromatography (HPLC). To evaluate the biological activities (an-tioxidant and anti-inflammatory) of these flavonoids, their in vitro inhibitory activities against lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) generation, nitric oxide (NO) production, and prostaglandin E2 (PGE2) secretion were determined. We successfully identified three flavonoids, namely luteolin (1), luteolin-7-O-β-D-glucoside (2), and luteolin-7-O-β-D-glucuronide (3) by spectral analyses. Luteolin (1) at 20 μg/mL inhibited ROS generation, NO production, and PGE2 secretion by 48.6%, 61.28% and 12.10%, respectively, and luteolin-7-O-β-D-glucoside (2) inhibited NO and PGE2 generation by 67.03% and 20.82%, respectively. Luteolin (1) and luteolin-7-O-β-D-glucoside (2) showed similar anti-inflammatory activities; however, luteolin (1) was observed to be a stronger antioxidant. Besides antioxidant and anti-inflammatory activities, S. brachyotus extract containing luteolin (1) and lu-teolin-7-O-β-D-glucoside (2) is considered to possess diverse biological activities. The results indicate that S. brachyotus is an edible medicinal plant, which is believed to be significant resource of functional foods.