This study was aimed to investigate the chemical composition and biological activities of leaf and stem essential oils of Zanthoxylum acanthopodium DC. from Vietnam. Their chemical composition was analyzed by GC/MS. Antimicrobial activities were evaluated by microdilution broth assay. Anti‐inflammatory activity was evaluated by the ability to inhibit nitric oxide production in macrophage cells. Cytotoxic activity was evaluated using the sulforhodamine B assay on three human cancer cell lines. Forty‐four compounds were identified in the leaf oil, among which dehydroaromadendrane (23.4 %), (E)‐carpacin (17.6 %), 2‐tridecanone (12.2 %), and 9‐methyl‐2‐decanone (11.8 %) were the most abundant. The stem oil contained fifty‐five identified constituents, mainly γ‐gurjunene (51.1 %) and butyl acetate (11.8 %). Both oils exhibited inhibitory effects on three bacterial strains, namely S. aureus, E. coli, P. aeruginosa and a fungal strain C. albican, while showed insignificant effects on B. subtilis, L. fermentum, and S. enterica. Both oils showed weak NO production inhibition in LPS‐induced RAW264.7 cells, but exhibited potent cytotoxic activity against all three tested cell lines SK‐LU‐1, MCF‐7, and HepG2 with the IC50 values ranging from 16.03±0.77 to 35.60±1.62 μg/mL. This is the first report on the antimicrobial, anti‐inflammatory and cytotoxic activities of essential oils from the leaves and stems of Z. acanthopodium.