Abstract: The antimicrobial activities and virulence intensity of the flavonoids which extracted with acetone, ethanol and water from Pinus massoniana needles were studied. And their qualitative analysis was examined by color development reaction, thin-layer chromatography, and infrared spectrum andcolorimetric method.Results showed that the 75% acetone extracts exhibited strongest antimicrobial activities to food spoilage bacteria, the diameter of inhibition zone was above 18 mm. The EC50 values of the falconoids for Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa and Salmonella paratyphi were18.5236, 43.8153, 36.5882, 33.0075, 28.2322, 14.4666 and 22.1227 mg /mL, respectively. The antibacterial activity of extracts was stabile to heat treatment and ultraviolet irradiation. Color development reaction, thin- layer chromatography and infrared spectrum were both showed that flavonoids were the main components in acetone extract from Pinus massoniana needles and the quality of flavonoids was 4.218 mg /mL.