Chemical composition, antimicrobial, antioxidant, and cytotoxic properties of Actinidia arguta essential oil (AEO) were evaluated. Gas chromatography-mass spectrometry analysis identified 56 chemical compounds, with the most abundant being Squalene (23.08%), γ-sitrostorol (8.10%), and β-Tocopherol (7.01%). Whereas the AEO had significant antimicrobial activity against Staphylococcus aureus and Saccharomyces cerevisiae, it showed mild efficacy against Bacillus subtilis and Microsporum canis. On the contrary, the Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, were not susceptible to the AEO pressure. On the other hand, the AEO exhibited strong antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH), β-carotene, and hydroxyl radicals, with IC values of 117.60, 73.60, and 35.15 μg/mL, respectively. Additionally, compared to the PC-3 or HT-29 cell lines, the A549 cells were more susceptible to the AEO (IC; 6.067 mg/mL). Besides, the confocal laser scanning microscopy imaging showed that 16 mg/mL of the AEO-induced apoptosis in the A549 cell lines. Our data indicated that the AEO might be useful in the food and pharmaceutical industry. Preparation of Actinidia arguta essential oil (a) and schematic overview of the experiment (b).