Panax notoginseng and its flowers are both well-known traditional Chinese medicinal herbs. To date, antimicrobial and antioxidant activities of flavonoids from P. notoginseng flowers (PNF) remain unclear. In this study, antimicrobial and antioxidant activities of flavonoids from PNF were investigated. The crude flavonoids were purified using a column (25 cm × 1.5 cm) packed with AB-8 macroporous adsorption resin. Compared with ascorbic acid, the purified flavonoids excelled in scavenging activities on 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid, superoxide anion and hydroxyl radicals at 0.2–1.0 mg/mL concentration. However, flavonoids exhibited weaker reducing power than ascorbic acid at 20–100 μg/mL concentration. In addition, the flavonoids exhibited obvious inhibitory effects on Staphylococcus aureus, Aeromonas hydrophila and Pseudomonas aeruginosa. These results clearly indicate that flavonoids from PNF are effective in scavenging free radicals and have the potential to be used as antioxidants and antimicrobial agents, and also provide the theoretical data for supporting the use of PNF in food, pharmaceutical and cosmetics industries.