Aflatoxin B1 (AFB1) contamination of aquaculture feed poses serious health risks to fish and consumers. In this study, the efficacy of a novel anti-mycotoxin agent specific for aquatic species (AM-AQ) containing bentonite, sepiolite, and orange peel meal (Citrus Sinensis) in alleviating AFB1 toxicity in Nile tilapia (Oreochromis niloticus) was evaluated. Fish were fed a basal diet as a control group and diets containing AFB1 (50 μg/kg), AM-AQ (3 g/kg), or a combination of both for 6 weeks. AFB1 exposure significantly reduced growth, altered hematological parameters, protein profile, and digestive enzyme activities (p < 0.05), and suppressed respiratory burst activity (p < 0.001). It also altered the expression of liver and kidney function enzymes, lysozyme, and pro-inflammatory cytokines and generated oxidative stress (p < 0.05, p < 0.01). AM-AQ supplementation effectively counteracted these adverse effects by restoring these parameters to control levels. Notably, AM-AQ reduced AFB1 residues in the musculature by 99.6% after 6 weeks (p < 0.001). Histopathological analysis revealed severe liver, spleen, and intestinal damage in the group exposed to AFB1, which was markedly ameliorated by AMAQ treatment (p < 0.05). These results demonstrate that the AM-AQ is a promising feed additive for protecting Nile tilapia against AFB1 toxicity and for reducing AFB1 transfer to edible tissues. Further research is required to optimize its application in aquaculture.