The aim of the present study was to evaluate the effect of a commercial Bacillus direct-fed microbial (DFM) on aflatoxin B1 toxic effects, performance, and biochemical and immunologic parameters in broiler chickens. Ninety 1-day-old Cobb 500 male broiler chicks were raised in floor pens for a period of 21 days. Chicks were neck-tagged, individually weighed, and randomly allocated to one of three groups: Negative control (basal feed), aflatoxin B1 (basal feed + 2 ppm AFB1), and DFM (basal feed + 2 ppm AFB1 + Bacillus direct-fed microbial). Each group had three replicates of 10 chickens (n = 30/group). Body weight and body weight gain were calculated weekly, while feed intake and feed conversion ratio were determined when broilers were 21 days old. On day 21, all chickens were bled, gastrointestinal samples were collected, and spleen and bursa of Fabricius were weighed. This study confirmed that 2 ppm of AFB1 causes severe detrimental effects on performance, biochemical parameters, and immunologic parameters, generating hepatic lesions in broiler chickens (P < 0.05). However, it was also observed that DFM supplementation provided beneficial effects that might help to improve gut barrier function, anti-inflammatory and antioxidant activities, as well as humoral and cellular immunomodulation. The results of the present study suggest that this Bacillus-DFM added at a concentration of 10⁶ spores/gram of feed can be used to counteract the negative effects that occur when birds consume diets contaminated with AFB1, showing beneficial effects on performance parameters, relative organ weights, hepatic lesions, immune response, and serum biochemical variables. The addition of this Bacillus-DFM might mitigate and decrease aflatoxicosis problems in the poultry industry, improving food security, alleviating public health problems, and providing economic benefits. Future studies are needed to fully elucidate the specific mechanisms by which this Bacillus-DFM counteracts the toxic effects of aflatoxin B1.