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The current study aimed to examine the in vitro and in vivo antifungal potential of pinocembrin-7-glucoside (P7G). P7G is an antifungal flavanone glycoside isolated from <i>Ficus hirta</i> Vahl. fruit against <i>Penicillium italicum</i>, a causative pathogen of blue mold disease in citrus fruit, and this study elucidates its possible action mechanism. P7G had a prominent mycelial growth inhibitory activity against <i>P. italicum</i>, with an observed half maximal effective concentration, minimum inhibitory concentration and minimum fungicidal concentration of 0.08, 0.2, and 0.8 g/L, respectively. The data from the in vivo test show that P7G significantly reduced blue mold symptoms and disease development of <i>P. italicum</i> in artificially inoculated “Newhall” navel orange. Compared to the control, increases in the cell membrane permeability of <i>P. italicum</i> supernatant and decreases in the intracellular constituent (e.g., soluble protein, reducing sugar, and total lipid) contents of <i>P. italicum</i> mycelia were identified, supporting scanning electron microscopy and transmission electron microscopy observations. Furthermore, a marked decline in both chitin and glucanase contents of <i>P. italicum</i> mycelia treated with P7G was induced by increasing its related degrading enzyme activities, suggesting that the cell wall structure was destroyed. The current study indicated that P7G may be a novel alternative for reducing blue mold by suppressing mycelial growth of <i>P. italicum</i> via a cell membrane/wall-targeting mechanism.