Ultrasonic-assisted extraction of flavonoids from Panax notoginseng flowers (PNF) and its antimicrobial activities were first investigated. Single-factor and orthogonal experiment were used to investigate the optimum extraction condition. The results showed that, the combination of 90% ethanol, material-to-liquid ratio of 1:20 (g/mL), 1 h ultrasonic extraction and 3 times of extraction were optimal extraction condition with the highest yields of flavonoids from PNF at 40 kHz/200 W. Under the optimal extraction condition, the extraction rate was 2.49%. Also, flavonoids from PNF had obvious inhibitory effects on Staphylococcus aureus, Aeromonas hydrophila and Pseudomonas aeruginosa. These results clearly indicate that flavonoids from PNF have the potential to be antimicrobial agents, and also provide the theoretical data for supporting the use of PNF for food, pharmaceutical, cosmetics or nutraceutical industries.