Sour rot, caused by Geotrichum citri-aurantii, is an important potentially devastating postharvest disease, which cannot be controlled efficiently by any registered fungicides such as imazalil, thiabendazole and fludioxonil. This study investigated the antifungal activity and mechanisms of three species of insect antimicrobial peptides (i.e., thanatin, ponericin W1, and mastoparan-S) against G. citri-aurantii. The data showed that they could kill conidia and inhibit the mycelial growth of G. citri-aurantii in vitro. Ponericin W1 showed high hemolytic activity, however, thanatin and mastoparan-S showed negligible hemolytic activity. In addition, the three peptides could also effectively control citrus fruit decay. Scanning electron microscopy showed that the morphology of the hyphae was altered after treatment with peptides. The membrane integrity disruption after treatment with peptides was determined by extracellular conductivity, the release of cytoplasmic constituents and fluorescence microscopy assay. Gel retardation assay demonstrated that G. citri-aurantii DNA was a target of thanatin. These results suggested that the three peptides could be potential alternatives to fungicides to prevent sour rot in citrus fruit.