Corynespora cassiicola is the pathogen that causes Corynespora leaf fall (CLF) disease. Cassiicolin (Cas), a toxin produced by C. cassiicola, is responsible for CLF disease in rubber trees (Hevea brasiliensis). Currently, the molecular mechanism of the cytotoxicity of Cas and its host selectivity have not been fully elucidated. To gain insight into these issues, we analyzed the binding of Cas1 and Cas2 to membranes consisting of different plant lipids and their membrane-disruption activities. Our real-time observations with high-speed atomic force microscopy (HS-AFM) and confocal microscopy reveal that the binding and disruption activities of Cas1 and Cas2 are strongly dependent on the types of membrane lipids. The mixtures of DPPC with DPPA, MGDG, DGDG, and stigmasterol are more susceptible to membrane damage caused by Cas1 and Cas2 than DPPC alone or its mixtures with sitosterol, DGTS-d9, and DGTS. This difference derives from the stronger binding of the toxins to membranes with the former lipid composition. Cytotoxicity tests on rubber leaves of RRIV 1, RRIV 4, and PB 255 clones suggest that the toxins cause necrosis of rubber leaves, except for the strong resistance of PB 255 against Cas2. Cryo-SEM analyses of necrotic leaf tissues exposed to Cas1 confirm that cytoplasmic membranes are vulnerable to the toxin. Thus, the host selectivity of Cas toxin in CLF disease is attained by the lipid-dependent binding activity of Cas to the membrane, and the cytotoxicity of Cas arises from its ability to disrupt membranes.