Aspergillus flavus is a saprophytic fungus that contaminates crops with carcinogenic aflatoxin. In the present work, the antifungal effects of volatile organic compounds (VOCs) from Streptomyces alboflavus TD-1 against A. flavus were investigated. VOCs from 8-day-old wheat bran culture of S. alboflavus TD-1 displayed strong inhibitory effects against mycelial growth, sporulation, and conidial germination of A. flavus. Severely misshapen conidia and hyphae of A. flavus were observed by scanning electron microscopy after exposure to VOCs for 6 and 12 h, respectively. Rhodamine 123 staining of mitochondria indicated that mitochondria may be a legitimate antifungal target of the VOCs from S. alboflavus TD-1. Furthermore, the VOCs effectively inhibited aflatoxin B1 production by downregulating genes involved in aflatoxin biosynthesis. Dimethyl trisulfide and benzenamine may play important roles in the suppression of A. flavus growth and production of aflatoxin. The results indicate that VOCs from S. alboflavus TD-1 have tremendous potential to be developed as a useful bio-pesticide for controlling A. flavus.