Tuberatolide B (TTB, C27H34O4) is a diastereomeric meroterpenoid isolated from the Korean marine algae Sargassum macrocarpum. However, the anticancer effects of TTB remain unknown. In this study, we demonstrate that TTB inhibits tumor growth in breast, lung, colon, prostate, and cervical cancer cells. To examine the mechanism by which TTB suppresses cell growth, we determined the effect of TTB on apoptosis, ROS generation, DNA damage, and signal transduction. TTB induced ROS production in MDA-MB-231, A549, and HCT116 cells. Moreover, TTB enhanced DNA damage by inducing γH2AX foci formation and the phosphorylation of DNA damage-related proteins such as Chk2 and H2AX. Furthermore, TTB selectively inhibited STAT3 activation, which resulted in a reduction in cyclin D1, MMP-9, survivin, VEGF, and IL-6. In addition, TTB-induced ROS generation caused STAT3 inhibition, DNA damage, and apoptotic cell death. Therefore, TTB suppresses cancer progression by promoting ROS-mediated inhibition of STAT3 signaling, suggesting that TTB is useful for the treatment of cancer.