Although aflatoxin M₁ (AFM₁) is the only mycotoxin with an established maximum residue limit (MRL) in milk worldwide, it’s common to find the co-occurrence of AFM₁ and ochratoxin A (OTA) in milk. The gastrointestinal tract (GIT) is the first tissue barrier to come into contact with food contaminants, such as mycotoxins, and intestinal epithelial cells are most affected. The GIT barrier is constituted by intercellular tight junction (TJ) proteins that localize to the apical domain of epithelial cells. Aflatoxin M₁ (AFM₁) and ochratoxin A (OTA) are mycotoxins commonly found in milk; however, their effects on intestinal epithelial cells have not been reported. In the present study, we show that AFM₁ (0.12 and 12 μM) and OTA (0.2 and 20 μM) individually or collectively increased the paracellular flux of lucifer yellow and fluorescein isothiocyanate (FITC)-dextrans (4 and 40 kDa) and decreased transepithelial electrical resistance values in differentiated Caco-2 cells after 48 h of exposure, indicating increased epithelial permeability. Immunoblotting and immunofluorescent analysis revealed that AFM₁, OTA, and their combination decreased the expression levels of tight junction (TJ) proteins and disrupted their structures, namely, claudin-3, claudin-4, occludin, and zonula occludens-1 (ZO-1), and p44/42 mitogen-activated protein kinase (MAPK) partially involved in the mycotoxins-induced disruption of intestinal barrier. The effects of a combination of AFM₁ and OTA on intestinal barrier function were more significant (P < 0.05) than those of AFM₁ and OTA alone, yielding additive or synergistic effects. The additive or synergistic effects of AFM₁ and OTA on intestinal barrier function might affect human health, especially in children, and toxin risks should be considered.