Marine sponge-derived manoalide has a potent anti-inflammatory effect, but its potential application as an anti-cancer drug has not yet been extensively investigated. The purpose of this study is to evaluate the antiproliferative effects of manoalide on oral cancer cells. MTS assay at 24 h showed that manoalide inhibited the proliferation of six types of oral cancer cell lines (SCC9, HSC3, OC2, OECM-1, Ca9-22, and CAL 27) but did not affect the proliferation of normal oral cell line (human gingival fibroblasts (HGF-1)). Manoalide also inhibits the ATP production from 3D sphere formation of Ca9-22 and CAL 27 cells. Mechanically, manoalide induces subG1 accumulation in oral cancer cells. Manoalide also induces more annexin V expression in oral cancer Ca9-22 and CAL 27 cells than that of HGF-1 cells. Manoalide induces activation of caspase 3 (Cas 3), which is a hallmark of apoptosis in oral cancer cells, Ca9-22 and CAL 27. Inhibitors of Cas 8 and Cas 9 suppress manoalide-induced Cas 3 activation. Manoalide induces higher reactive oxygen species (ROS) productions in Ca9-22 and CAL 27 cells than in HGF-1 cells. This oxidative stress induction by manoalide is further supported by mitochondrial superoxide (MitoSOX) production and mitochondrial membrane potential (MitoMP) destruction in oral cancer cells. Subsequently, manoalide-induced oxidative stress leads to DNA damages, such as γH2AX and 8-oxo-2’-deoxyguanosine (8-oxodG), in oral cancer cells. Effects, such as enhanced antiproliferation, apoptosis, oxidative stress, and DNA damage, in manoalide-treated oral cancer cells were suppressed by inhibitors of oxidative stress or apoptosis, or both, such as N-acetylcysteine (NAC) and Z-VAD-FMK (Z-VAD). Moreover, mitochondria-targeted superoxide inhibitor MitoTEMPO suppresses manoalide-induced MitoSOX generation and γH2AX/8-oxodG DNA damages. This study validates the preferential antiproliferation effect of manoalide and explores the oxidative stress-dependent mechanisms in anti-oral cancer treatment.