Abstract13‐Acetoxysarcocrassolide (13‐AC) is a marine cembranoid derived from the aquaculture soft coral of Lobophytum crassum. The cytotoxic effect of 13‐AC against leukemia cells was previously reported but its mechanism of action is still unexplored. In the current study, we showed that 13‐AC induced apoptosis of human acute lymphoblastic leukemia Molt4 cells, as evidenced by the cleavage of PARP and caspases, phosphatidylserine externalization, as well as the disruption of mitochondrial membrane potential. The use of N‐acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, attenuated the cytotoxic effect induced by 13‐AC. Molecular docking and thermal shift assay indicated that the cytotoxic mechanism of action of 13‐AC involved the inhibition of heat shock protein 90 (Hsp 90) activity by eliciting the level of Hsp 70 and topoisomerase IIα in Molt4 cells. 13‐AC also exhibited potent antitumor activity by reducing the tumor volume (48.3%) and weight (72.5%) in the in vivo Molt4 xenograft mice model. Our findings suggested that the marine cembranoid, 13‐AC, acted as a dual inhibitor of Hsp 90 and topoisomerase IIα, exerting more potent apoptotic activity via the enhancement of ROS generation.