IN tropical and subtropical regions, contamination of crops by aflatoxin B1 (AFB1) is a growing challenge. To restrict fungal growth and allow aflatoxin detoxification, there is a need to control mycotoxin contamination. Streptomyces exfoliatus has shown promise against several phytopathogenic fungi. This study aimed to explore whether S. exfoliatus could be employed as a biocontrol agent against Aspergillus flavus and AFB1 contamination. In this study, the biological control of AFB1 production by A. flavus was examined using some actinobacteria isolates. The cytotoxic activity of AFB1 and its degradation products were also evaluated. Results revealed that the most effective actinobacterium against A. flavus was isolate number 1, which was identified as S. exfoliatus. The growth, sporulation, and AFB1 production of A. flavus decreased when it was treated with a cell-free culture filtrate produced by the identified isolate. Furthermore, 20% of the cell-free culture filtrate of S. exfoliatus completely inhibited AFB1 production. The AFB1 content was also reduced in wheat samples treated with day intervals. After 21 days, no AFB1 was detected compared with those of the controls. After 3 days, 95.47% of AFB1 was degraded by the cell-free culture filtrate. The degraded AFB1 products were less toxic than the parent aflatoxin. The optimum temperature of AFB1 degradation was 30°C, but AFB1 degradation decreased as the temperature further increased. The cell-free culture filtrate remained stable for 12 months at the time of freezing. In conclusion, S. exfoliatus cell-free culture filtrates can inhibit the growth and sporulation of A. flavus as well as reduce AFB1 generation and degrade it into less harmful compounds. This appears a promising option for minimizing contamination by A. flavus, preventing aflatoxin accumulation, and enabling aflatoxin breakdown in wheat grains; this approach may be used to reduce A. flavus and AFB1 contamination in future biological control programs.