The antifungal activity, stability and mechanism of limonin from lemon peel against Aspergillus niger were investigated. The results showed that the minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) of limonin from lemon peel on Aspergillus Niger were 625 μg/mL and 5000 μg/mL respectively, and the half-effective concentration (EC50) on spore germination and mycelium growth were 311.03 μg/mL and 291.63 μg/mL respectively. The antifungal activity of limonin on Aspergillus niger was weakened under UV light and pH 6~7. Fe3+ and Fe2+ could enhance the antifungal activity. The limonin from lemon peel could greatly change extracellular alkaline phosphatase (AKP) activity, soluble protein and total sugar content of mycelium of Aspergillus niger. Therefore, limonin destroyed the integrity of cell wall and cell membrane of Aspergillus niger, resulting in a large amount of content exudation, affecting the normal metabolism of cells, thereby inhibiting the growth and reproduction of cells.