Penicillium italicum is the causal agent of citrus blue mold, which is a major threat to the global citrus fruit industry. Antofine, a natural phenanthroindolizidine alkaloid, is water-soluble and exhibits a broad range of biological activities. However, whether it can inhibit P. italicum growth and the potential inhibitory mechanism remains to be elucidated. This study aimed to investigate the antifungal mechanism of antofine against P. italicum using scanning electron microscopy, transmission electron microscopy (TEM), propidium iodide staining, and tandem mass tag-labeled quantitative proteomic analysis. Antofine was found to exhibit its preeminent antifungal activity against P. italicum with a minimum inhibitory concentration of 1.56 mg/L and a minimum fungicidal concentration of 6.25 mg/L. The challenge test revealed that antofine inhibited the development of citrus blue mold during a 6-d P. italicum-infected period. Antofine acted on its potential multitargets to inhibit P. italicum growth by synergistically activating oxidative stress through accumulating excess reactive oxygen species, impairing membrane integrity, inducing membrane lipid peroxidation, and disrupting mitochondrial function, thereby disrupting the membrane system and reducing cell viability. Moreover, antofine treatment downregulated most differentially expressed proteins involved in carbon metabolism, pyruvate metabolism, and the tricarboxylic acid cycle (TCA) in P. italicum mycelia, which may explain the mitochondrial decomposition observed by TEM and the declines in ATP levels as well as the activities of TCA-related enzymes. These results indicate that antofine treatment inhibited P. italicum growth by targeting the cell membrane and mitochondria. © 2023 The Author(s).