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Chrysanthemum morifolium cv. Hang-ju (HJ) is considered as a healthy food supplement for a long time in Asia. But until now, there are few reports on the biological activity of HJ leaves, which is the main by-products of HJ. In this study, HJ leaves was extracted by water, ethanol and water: ethanol (30: 70), respectively. The results showed that the total polyphenol content of water extract, hydroalcoholic extract and ethanol extract was 75.1 ± 6.9 mg GAE/g, 162.2 ± 5.6 mg GAE/g and 128.5 ± 1.9 mg GAE/g, respectively. However, the ethanol extract has the highest antibacterial activity against the tested strains with a MIC of 0.25 mg/mL compared to water extract (MIC > 8.0 mg/mL) and ethanol extract (MIC = 0.50 mg/mL). The essential oil was extracted from HJ leaves by water distillation (0.5%), which showed higher antibacterial effect on gram-positive bacteria (MIC = 0.125 mg/mL) than ethanol extract. GC–MS was used for the preliminary identification of essential oil composition. The compound (1S,4aS,7R,8aS)-1,4a-dimethyl-7-(prop-1-en-2-yl)decahydronaphthalen-1-ol was first identified from HJ and found to be the most dominant compound. Furthermore, essential oil can significantly deform cell morphology and damage cell wall structure, which was confirmed by LIVE/DEAD® BacLight Viability Kit and scanning electron microscopy observations. The mentioned findings indicated that HJ leaves could be utilized as a natural additive in food industry with significant antimicrobial potential. [ABSTRACT FROM AUTHOR]