Overstimulation of CFTR-mediated Cl− secretion plays an important role in the pathogenesis of secretory diarrheas, which remain an important global health problem. This study aimed to identify inhibitors of CFTR-mediated Cl− secretion from a library of fungus-derived compounds and to evaluate their pharmacological properties and anti-diarrheal utility. We identified zearalenone, 7′-dehydrozearalenone and 8′-hydroxyzearalenone isolated from the seagrass-derived fungus Fusarium sp. PSU-ES123 as inhibitors of CFTR-mediated Cl− secretion in human intestinal epithelial (T84) cells. Being the most potent fungal metabolite capable of inhibiting CFTR-mediated Cl− secretion, zearalenone reversibly inhibited CFTR Cl− channel activity in T84 cells with an IC50 of ˜0.5 μM. Functional and biochemical analyses and molecular docking studies indicate that zearalenone binds to the β-estradiol binding sites in the ATP-binding pockets on NBD1 and NBD2 of CFTR. Mechanisms of CFTR inhibition by zearalenone do not involve activation of phosphodiesterases, protein phosphatases, multidrug-resistance protein 4 and AMP-activated protein kinases. Importantly, zearalenone significantly inhibited cholera toxin (CT)-induced Cl− secretion in T84 cells and blocked CT-induced intestinal fluid secretion in mice. Collectively, our study indicates that zearalenone represents the first class of fungus-derived CFTR inhibitors. Further development of this class of compounds may provide an effective treatment of secretory diarrheas.