The human intestine is regularly exposed to ingested food contaminants, such as fungal and bacterial toxins, which have been described to co-occur in a mixed diet. Thus, it is of utmost importance to understand possible interactions between contaminants of different origin. Hence, we investigated the single and combined effects of one of the most abundant mycotoxins, deoxynivalenol (DON; 0.1 to 10 µg/mL), and the bacterial toxin cereulide (CER; 1 to 100 ng/mL) on differentiated human Caco-2 (C2BBe1) cells cultured in a transwell system. We tested the capacity of the two toxins to alter the intestinal integrity and further investigated the uptake of both compounds and the formation of selected DON metabolites. CER alone (10 and 100 ng/mL) and in combination with DON (10 ng/mL CER with 1 µg/mL DON) was found to alter the barrier function by increasing the transepithelial electrical resistance and the expression of the tight junction protein claudin-4. For the first time, DON-3-sulfate was identified as a metabolite of human intestinal cells in vitro. Moreover, co-incubation of CER and DON led to an altered ratio between DON and DON-3-sulfate. Hence, we conclude that co-exposure to CER and DON may alter the intestinal barrier function and biotransformation of intestinal cells.