Postharvest diseases, such as black spots caused by Alternaria alternata, 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 W. anomalus, 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 W. anomalus on tomato black spot disease and the inhibition effect of main components on A. alternata. A GC-MS analysis found that isoamyl acetate was the main component of W. anomalus that played an inhibitory role. The results showed that isoamyl acetate could inhibit the growth of A. alternata 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.