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• Ultrasound pretreatment combined with microwave-assisted hydrodistillation is used. • Ultrasound and microwave factors affect the kinetics and essential oil composition. • First-order kinetic model is suitable for the microwave isolation of essential oil. • Essential oil exerts higher antioxidant, antifungal and cytotoxic activity. Plant essential oil is an important resource of natural medicinal products, thus development of efficient techniques for the isolation of essential oils is one of the research hotspots. In this work, ultrasound was applied to pre-treat Perilla frutescens (L.) Britt. leaves for the following microwave-assisted hydrodistillation (UP-MAHD) of essential oils. The influence of ultrasound pre-treatment and microwave-assisted hydrodistillation procedure factors on the kinetics and essential oil composition were investigated. Additionally, the kinetic curves under different conditions were fitted using models, which revealed that the first-order kinetic model was more suitable. Compared with reference techniques, the ultrasound pre-treatment procedure could significantly improve the yield of essential oil and the percentage of oxygenated components, which might be attributed to the synergy between ultrasound and microwave irradiation. Gas chromatography-mass spectrometer analyzed that the essential oils were rich in perilla ketone (80.76%), other major components included β-caryophyllene (1.65%), linalool (1.15%), caryophyllene oxide (1.12) and apiol (1.19%). The essential oils from UP-MAHD showed higher antioxidant activity (IC 50 = 2.60 μL/mL). Moreover, Colletotrichum gloeosporioides and Fusarium fujikuroi were two of the most sensitive fungal strains to essential oils. As compared with paclitaxel, essential oils presented better cytotoxic activity against human gastric cancer cell (MGC-803) and lung cancer cell (A549). Overall, the developed technique remarkably improves the yield of essential oil and its biological activity, which is a promising alternative and provides a foundation for the efficient utilization of P. frutescens resource and other crops. [ABSTRACT FROM AUTHOR]