Deoxynivalenol (DON) and fumonisins (FBs) are secondary metabolites produced by Fusarium fungi which frequently contaminate broiler feed. The aim of this study was to investigate the impact of DON and/or FBs on the intestinal barrier in broiler chickens, more specifically on the mucus layer and antioxidative response to oxidative stress. One-day-old broiler chicks were divided into four groups, each consisting of eight pens of seven birds each, and were fed for 15 days either a control diet, a DON contaminated diet (4.6 mg DON/kg feed), a FBs contaminated diet (25.4 mg FB1+FB2/kg feed), or a DON and FBs contaminated diet (4.3 mg DON and 22.9 mg FB1+FB2/kg feed). DON and FBs affected the duodenal mucus layer by suppressing the intestinal mucin (MUC) 2 gene expression and altering the mucin monosaccharide composition. Both mycotoxins decreased gene expression of the intestinal zinc transporter (ZnT)-1, and regulated intracellular methionine homeostasis, which are both important for preserving the cell's critical antioxidant activity. Feeding a DON and/or FBs contaminated diet, at concentrations close to the European Union maximum guidance levels (5 mg DON and 20 mg FB1+FB2/kg feed) changes the intestinal mucus layer and several intestinal epithelial antioxidative mechanisms.