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Aflatoxin B1 (AFB1) and ochratoxin A (OTA) naturally co-occur in several foods, but no studies have followed the fate of mycotoxins’ interactions along the gastrointestinal tract using in vitro digestion models. This study used a novel semi-dynamic model that mimics gradual acidification and gastric emptying, coupled with a static colonic fermentation phase, in order to monitor mycotoxins’ bioaccessibility by the oral route. AFB1 and OTA bioaccessibility patterns differed in single or co-exposed scenarios. When co-exposed (MIX meal), AFB1 bioaccessibility at the intestinal level increased by ~16%, while OTA bioaccessibility decreased by ~20%. Additionally, a significant increase was observed in both intestinal cell viability and NO production. With regard to mycotoxin–probiotic interactions, the MIX meal showed a null effect on Lactobacillus and Bifidobacterium strain growth, while isolated AFB1 reduced bacterial growth parameters. These results were confirmed at phylum and family levels using a gut microbiota approach. After colonic fermentation, the fecal supernatant did not trigger the NF-kB activation pathway, indicating reduced toxicity of mycotoxins. In conclusion, if single exposed, AFB1 will have a significant impact on intestinal viability and probiotic growth, while OTA will mostly trigger NO production; in a co-exposure situation, both intestinal viability and inflammation will be affected, but the impact on probiotic growth will be neglected.