Abstract: Microbial contamination significantly compromises the commercial value and food safety of raisins. Controlling mold proliferation is essential to prevent spoilage and to preserve fruit quality. This study evaluated the antifungal efficacy and underlying mechanisms of natamycin against the primary spoilage fungi associated with seedless white raisins: Aspergillus tubingensis, Aspergillus niger, and Penicillium steckii. Natamycin at a concentration of 900 mg/L completely inhibited visible fungal spoilage caused by all three species, with inhibitory effects increasing dose-dependently. The minimum inhibitory concentrations (MIC) for the three molds were determined to be 180, 220, and 220 mg/L, respectively. At 1MIC, natamycin significantly (P<0.05) reduced spore germination rates, germ tube lengths, and overall mycelial growth in all tested fungi. Scanning electron microscopy revealed pronounced morphological abnormalities, including twisted and collapsed hyphae. Furthermore, natamycin treatment led to significant (P<0.05) incraese in relative conductivity and leakage of intracellular contents, indicating compromised cell membrane integrity. Ergosterol levels decreased markedly (P<0.05), while malondialdehyde (MDA) content increased significantly (P<0.05), suggesting oxidative stress and menbrane lipid peroxidation. Collectively, these findings demonstrated that natamycin effectively inhibited fungal growth and spoilage in seedless white raisins by disrupting membrance integrity and interfering with key physiological processess.