Abstract In this work, the nanoemulsion of essential oil from Citrus medica L. var. sarcodactylis was prepared using a spontaneous emulsification method. Meanwhile, the effect of nanoemulsification on the antioxidant, antibacterial and antibiofilm activity of essential oil was evaluated. GC–MS analysis indicated nineteen compounds in the essential oil, and the essential oil was dominated by d -Limonene (50.04%). The minimum and stable droplets (73 nm) of essential oil nanoemulsion were formulated when the concentration of essential oil in oil phase was 50% (w/w). The hydroxyl radicals, DPPH radicals scavenging ability, iron reducing power, antibacterial and antibiofilm activity of nanoemulsified essential oil were significantly higher than those of pure essential oil at same concentration. Moreover, the nanoemulsified essential oil exhibited higher inhibitive effect on the bacteria in tofu than pure essential oil. Therefore, nanoemulsification significantly enhanced the antioxidant, antibacterial and antibiofilm activity/efficiency of essential oils, which will promote the abroad utilization of essential oils.