This study examined the inhibitory effects of Iturin A on food spoilage yeasts and tried to elucidate the underlying mechanisms of its action. Four yeast strains—Saccharomyces cerevisiae, Pichia kluyveri, Debaryomyces hansenii, and Cyberlindnera fabianii—were evaluated through inhibition zone diameters, minimum inhibitory concentration, and minimum fungicidal concentration. Iturin A effectively inhibited all four strains, with minimum inhibitory concentration, and minimum fungicidal concentration of 0.03 and 0.06 g/L, 0.06 and 0.125 g/L, 0.125 and 0.50 g/L, and 0.125 and 0.25 g/L, respectively. Cell permeability assays demonstrated that Iturin A increased membrane permeability, resulting in nucleic acid and protein leakage. Scanning electron microscopy altered cell morphology, suggesting membrane damage following Iturin A treatment. Non-targeted metabolomic analysis indicated that Iturin A exerts its antifungal effects through multiple mechanisms, including disruption of fatty acid and phospholipid profiles in the cell membrane, impairment of ion channels, and cell wall disruption via inhibition of glycosylphosphatidylinositol-anchored molecule synthesis. Additionally, Iturin A induced DNA damage, which prompted S. cerevisiae cells to enhance uridine 5′-diphosphate synthesis, promote cell wall and DNA synthesis, elevate DNA methylation, activate purine salvage pathway, promote DNA repair, and strengthen the tricarboxylic acid cycle to enhance energy production. These findings offer novel insights into the antifungal mechanisms of Iturin A and underscore its potential for controlling food spoilage.