Abstract Purpose: The DEAD-box RNA helicase eIF4A1 carries out the key enzymatic step of cap-dependent translation initiation and is a well-established target for cancer therapy, but no drug against it has entered evaluation in patients. We identified and characterized a natural compound with broad antitumor activities that emerged from the first target-based screen to identify novel eIF4A1 inhibitors. Experimental Design: We tested potency and specificity of the marine compound elatol versus eIF4A1 ATPase activity. We also assessed eIF4A1 helicase inhibition, binding between the compound and the target including binding site mutagenesis, and extensive mechanistic studies in cells. Finally, we determined maximum tolerated dosing in vivo and assessed activity against xenografted tumors. Results: We found elatol is a specific inhibitor of ATP hydrolysis by eIF4A1 in vitro with broad activity against multiple tumor types. The compound inhibits eIF4A1 helicase activity and binds the target with unexpected 2:1 stoichiometry at key sites in its helicase core. Sensitive tumor cells suffer acute loss of translationally regulated proteins, leading to growth arrest and apoptosis. In contrast to other eIF4A1 inhibitors, elatol induces markers of an integrated stress response, likely an off-target effect, but these effects do not mediate its cytotoxic activities. Elatol is less potent in vitro than the well-studied eIF4A1 inhibitor silvestrol but is tolerated in vivo at approximately 100× relative dosing, leading to significant activity against lymphoma xenografts. Conclusions: Elatol's identification as an eIF4A1 inhibitor with in vivo antitumor activities provides proof of principle for target-based screening against this highly promising target for cancer therapy. Clin Cancer Res; 24(17); 4256–70. ©2018 AACR.