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Postharvest diseases, such as black spots caused by <i>Alternaria alternata</i>, have caused huge economic losses to the tomato industry and seriously restricted its development. In recent years, biological control has become a new method to control postharvest diseases of fruits and vegetables. Our research group screened <i>W. anomalus</i>, a yeast demonstrating a promising control effect on a postharvest black spot disease of tomatoes, and explored its physiological mechanism of prevention and control. Therefore, this study investigated the prevention and control effect of metabolites of <i>W. anomalus</i> on tomato black spot disease and the inhibition effect of main components on <i>A. alternata</i>. A GC-MS analysis found that isoamyl acetate was the main component of <i>W. anomalus</i> that played an inhibitory role. The results showed that isoamyl acetate could inhibit the growth of <i>A. alternata</i> and had a certain control effect on postharvest black spots in tomatoes. Our findings suggest that isoamyl acetate could be a promising alternative to fungicides for controlling postharvest black spots in tomatoes.