This study was aimed at determining the cytotoxicity and antimigration activity of zerumbone isolated fromZ. ottensii Val. rhizomes against HeLa cell lines. Zerumbone was isolated from the hexane fraction of the ethanol extract by a bioassay-guided procedure using column chromatography, and spectroscopic data analysis was usedtoestablish its structure. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was usedtomeasure zerumbone's cytotoxic activity. The inhibition of zerumbone against HeLa cell migration was assessedusing the scratch/wound healing test. DHODH inhibition activity determined by molecular docking usingAutoDock 4.2.6 and in vitro biochemical assay. The results of the MTT assay showed that zerumbone hadhighcytotoxicity against HeLa cell lines, with the IC50 value of 41.60 µg/mL in the 24-hour treatment. According tothecell migration assay, zerumbone suppressed HeLa cell migration in both the 24 and 48-hour treatments inaconcentration-dependent manner. Zerumbone at a concentration of 15 µg/mL significantly inhibited the migrationof the HeLa cells compared with the control. Zerumbone has good affinity with the DHODH enzyme and has potential activity as a DHODH inhibitor. This study indicates that zerumbone of the Z. ottensii rhizome has potential tobeexplored further as an anticancer drug candidate.