Abstract Milk is a key food worldwide prone to mycotoxins contamination. Lactobacillus plantarum and prebiotics detoxification ability was evaluated by a Plackett-Burman Design considering the reduction of aflatoxin B1 (AFB1) and its bioaccessibility in artificially contaminated ultra-high temperature cow milk. Six variables were evaluated: AFB1 concentration (from 5.0 to 10.0 μg L−1); incubation time (0 to 6 h); and inulin, oligofructose, β-glucan, and polydextrose concentrations (from 0.00 to 0.75%). The reduction in AFB1 ranged from 0% to 55.85% and in vitro bioaccessibility from 15.62% to 51.09%. The greatest reduction in AFB1 occurred by adding L. plantarum combined with inulin, oligofructose and β-glucan. The greatest reduction in bioaccessibility occurred by adding inulin or oligofructose and L. plantarum with a 10.0 μg L−1 AFB1 concentration. A sharp reduction in AFB1 was accompanied by higher bioaccessibility rates, and in this case, bioaccessibility is considered the main factor to ensure a low AFB1 absorption by the body. The best experimental condition was 10.0 µg L-1 AFB1, added of L. plantarum and inulin or oligofructose (0.75%), ensuring > 16% final bioaccessibility. Such results represent a safe AFB1 decontamination level for milk.