Objective To extract the potentially antimicrobial components from cortex of Broussonetia papyrifera. Methods The optimization of ultrasonic extraction of flavonoids from cortex of Broussonetia papyrifera was performed using the orthogonal design. The active components of flavonoids extract from cortex of Broussonetia papyrifera were enriched using AB-8 macroporous resin. Comparison of antimicrobial effects between different components was performed using drug-containing culture medium. Results The optimal extraction of total flavonoids from cortex of Broussonetia papyrifera was performed by 20-min ultrasonic extraction (300 W) using 10 volumes of 70% ethanol as a solvent. After enrichment and separation using AB-8 macroporous resin, the 70% ethanol elution fraction had good inhibitory effects on superficial fungi including Trichophyton rubrum and Candida albicans. Conclusion This extraction technique features simple operation, low power consumption, high extraction rate of total flavonoids, and potent antimicrobial activity of antimicrobial components.