The objectives of this work were to evaluate the antioxidant and antibacterial activities of peony flowers extract and to identify chemical constituents of the active chromatographic fraction. Ethyl acetate was used as a solvent for extraction. The crude flower ethyl acetate extract was further purified by column chromatography to yield four fractions. Each purified fraction was evaluated for the antioxidant and antibacterial activities. The optimal antioxidant activity found in fraction 2 (IC50 = 1.156±0.031 ppm), for DPPH assay, compared with standard ascorbic acid (3.086±0.070 ppm). Fraction 2 also showed the highest value of total phenolic content (TPC = 427.737±0.007 µg GAE/mg dry weight) and the greatest total flavonoid compound (TFC = 85.926±0.007 µg QE/mg dry weight). This fraction exhibited a high value of inhibition zone (2.156±0.027 cm for E. coli and 1.537±0.124 cm for S. aureus), compared with penicillin (positive control 1.612±0.053 cm for E. coli and 2.563±0.194 cm for S. aureus). It was subjected to liquid chromatography mass spectroscopy (LCMS) in order to identify the chemical constituents. The major compound was paeoninol with the m/z value of 679.6.