Phytochemical characteristics and antioxidant activities of the crude and fractionated plant extracts of Anoectochilus formosanus were evaluated using five different assay systems. An acid-treatment (2 N HCl in 95% ethanol) was employed to treat a butanol fraction (BuOH), creating an acid-hydrolyzed BuOH fraction. The IC(50) values for DPPH radicals in the BuOH and acid-hydrolyzed BuOH fractions were 0.521 and 0.021 mg/mL, respectively. The acid-hydrolyzed BuOH exhibited approximately 5-fold higher activity in scavenging superoxide anion than catechin. The acid-hydrolyzed BuOH fraction also effectively protected phi x174 supercoiled DNA against strand cleavage induced by H(2)O(2) and reduced oxidative stress in HL-60 cells. Metabolite profiling showed that the aglycones of flavonoid glycosides in BuOH were produced after acid hydrolytic treatment, and this resulted in a significant increase in antioxidant activities of acid-hydrolyzed BuOH. One new diarylpentanoid, kinsenone, and three known flavonoid glycosides and their derivatives were identified for the first time from A. formosanus, with strong antioxidant properties.