Guava (Psidium guajava L.) leaf essential oil (GLEO) was extracted by water distillation, and its in vitro antioxidant, antidiabetic, and antibacterial properties were evaluated. Using GC/MS to determine the chemical components of GLEO, 27 constituents were identified, accounting for 74.90 % of the total oil content, among which L-caryophyllene (24.46 %), L-calamenene (10.82 %), (À )-globulol (10.69 %), and α-copaene (8.71 %) were the main components. Subsequently, the antioxidant activity of GLEO was determined by DPPH, ABTS, and βcarotene bleaching tests. The half maximal inhibitory concentration of GLEO for three free radicals were IC 50 = 17.66 � 0.07 μg/mL, IC 50 = 19.28 � 0.03 μg/mL, and IC 50 = 3.17 � 0.01 μg/mL, respectively. Moreover, GLEO exhibited remarkable α-amylase (IC 50 = 13.99 � 0.34 μg/mL) and α-glucosidase (IC 50 = 5.50 � 1.02 μg/mL) inhibitory activities. It was effective against Streptomyces acidiscabies (MIC = 1.25 μg/mL), Ralstonia solanacearum (MIC = 5 μg/mL), and Erwinia carotovora subsp carotovora borgey (MIC = 2.5 μg/mL), showing significant antibacterial properties. Based on the findings, given the high biological activity of GLEO, it is a biological preservative for food, medicine, and cosmetics and is valuable in natural therapy and crop disease management.