Artemisia argyi Lévl. et Vant essential oil could be used as a good antimicrobial flavouring agent and applied in the food industry. In this study, three methods, including simultaneous distillation-extraction (SDE), subcritical extraction and hydrodistillation, were applied to extract A. argyi essential oil. Compared with subcritical extraction (1%) and hydrodistillation (0.5%), SDE gave a higher yield (1.2%). Components of the essential oils were analysed with gas chromatography-mass spectrometry (GC-MS), and the most abundant ingredients were caryophyllene oxide, neointermedeol, borneol, α-thujone and β-caryophyllene. These five components accounted for 82.93%, 40.90% and 40.33% for SDE, subcritical extraction, and hydrodistillation, respectively. Based on agar disc diffusion and minimum inhibitory concentration (MIC) assays, SDE oil showed a significant inhibitory effect towards Listeria monocytogenes, Escherichia coli, Proteus vulgaris, Salmonella enteritidis and Aspergillus niger. Furthermore, electron microscope observations (SEM) confirmed that SDE oil could obviously deform cell morphology and destroy the structure of cell walls. Performances showed that SDE was a promising process for extracting A. argyi essential oil with both high yield and antimicrobial activity.