ABSTRACT: The chemical constituents and the antioxidant, antimicrobial, and cytotoxic activities of fresh rhizome essential oil (FR-EO) and dry rhizome essential oil (DR-EO) of Zingiber zerumbet (L.) Smith obtained from Southwest China were compared. Zerumbone was the predominant component in both FR-EO and DR-EO (75.0% and 41.9%, respectively). FR-EO, DR-EO, and zerumbone were all demonstrated to have significant antimicrobial capacity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Proteus vulgaris, with minimum inhibitory concentration (MIC) ranging from 31.25 to 156.25??g/mL and minimum bactericidal concentration (MBC) ranging from 62.50 to 625.00??g/mL. Zerumbone showed the strongest antimicrobial potential against all tested microorganisms compared with the fresh and dry rhizome essential oils. FR-EO was found to be more active than DR-EO against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Proteus vulgaris. FR-EO, DR-EO, and zerumbone all showed significant cytotoxic activity against K562, PC-3, and A549 human tumor cell lines in a time- and concentration-dependent manner. Zerumbone exhibited the strongest antiproliferative activity against all tested human tumor cell lines with an IC50 of 4.21-11.09??g/mL for 72?h incubation, as compared with the fresh and dry rhizome oils. The cytotoxic activity of FR-EO (IC50: 10.48-14.51??g/mL for 72?h) was found to be significantly higher (p < 0.05) than that of DR-EO (IC50: 13.83-33.24??g/mL for 72?h). FR-EO, DR-EO, and zerumbone exhibited selective cytotoxic activity to tumor cells, with a significantly low cytotoxicity to normal cells (MRC-5, IC50: 56.98-147.29??g/mL). However, FR-EO, DR-EO, and zerumbone all exhibited weak free-radical-scavenging activity according to DPPH and ABTS analysis. The findings highlighted in this study show that FR-EO provides appreciably higher content of the bioactive compound, zerumbone, and has higher antimicrobial and cytotoxic properties than DR-EO. Thus, fresh Z. zerumbet rhizome should be preferred in cosmetic, food, and pharmaceutical applications.