Due to their potent antimicrobial activity, silver nanoparticles (AgNPs) synthesized by biomass might be promising in food preservation, whereas effects of AgNPs on Penicillium italicum-induced rot of Citrus fruits have not been investigated. In this study, a novel AgNPs biosynthesis method was developed based on Fatsia japonica leaf extracts. It was revealed that concentrations of leaf extracts, AgNO and NaCl affected AgNPs yields and particle sizes obviously. Under the optimized conditions (8 mg/mL extracts, 2 mM AgNO and 1 mM NaCl), AgNPs, synthesized within 80 min, showed potent preservative effect against P. italicum-induced rot of Citrus fruits. Furthermore, inhibition test and TEM analysis indicated that as-synthesized AgNPs caused cell deformation, cytoplasmic leakage, and thereupon cell death of P. italicum. Moreover, AgNPs had significant antibacterial activities against Escherichia coli and Staphylococcus aureus, which might be beneficial for Citrus fruits preservation. Altogether this study develops an efficient AgNPs synthesis method and a novel preservation method for Citrus fruits.