The volatile compositions and antimicrobial activity of aerial parts of Popowia pisocarpa from Vietnam were reported for the first time. From the GC/MS spectral, spathulenol (35.9%), bicyclogermacrene (5.7%) and muurola-4,10(14)-dien-1β-ol (4.2%) among the sesquiterpenoids, along with 4,4-dimethyl-3-(3-methylbut-3-enylidene)-2-methylenebicyclo[4,1,0]heptane (17.7%), an unsaturated cyclic compound, were the main constituents of the essential oil. Monoterpenoids were not identified in the essential oil. The essential oil displayed antimicrobial activity against the Gram-negative Escherichia coli ATCC 25922 with MIC value of 16.0 µg/mL and IC50 value of 8.52 µg/mL. In addition, the essential oil also exhibited activity towards the Gram-positive bacteria of Staphylococcus aureus ATCC 13709, Bacillus subtilis ATCC 6633, and Lactobacillus fermentum N4 with MIC value of 64.0 µg/mL each and corresponding IC50 values of 11.06 µg/mL, 26.47 µg/mL and 15.68 µg/mL, respectively. The essential oil did not inhibit the growth of Pseudomonas aeruginosa ATCC 15442, Salmonella enterica and the yeast Candida albicans ATCC 10231.