The bovine rumen is capable to detoxify certain mycotoxins to less toxic metabolites. However, during rumen disturbance e.g., sub-acute ruminal acidosis, the ability of detoxification might decrease. There is only a limited number of studies available evaluating the effect of mycotoxins on rumen and intestinal epithelium of cattle. The aim of the study was therefore to access the toxicity of 2 common mycotoxins: deoxynivalenol (DON) or fumonisin B₁ (FB₁) in primary bovine rumen epithelial cells (REC) and in a calf intestinal epithelial cell line (CIEB). REC were isolated from rumen tissue of dairy cows by enzymatic dissociation with trypsin. CIEB is a spontaneously immortalized cell line derived from the small intestine of a calf. Both cells types were characterized via immunostaining for cytokeratin (epithelial cell marker). For toxicity studies, cells were seeded in 96 well plates (2 × 10⁴ cells/well) for 24 h. Thereafter, cells were incubated with 0 to 25 µM DON and FB₁ (n = 6). After 48 h of incubation, the water-soluble tetrazolium salt (WST-1; Mitochondrial metabolism) assay, the neutral red (NR; Lysosomal Activity) assay as well as the sulforhodamine B (SRB; Total protein synthesis rate) assay were performed. Statistical evaluation of data was performed with GraphPad Prism software (Version 7). Analysis of variance or Kruskal-Wallis test was used for data evaluation, depending if data were normally distributed or not. Data were considered as significant if P < 0.05. REC as well as CIEB were positively stained for cytokeratin, and therefore confirmed as epithelial cells. DON had the greatest effect on mitochondrial metabolism in REC starting at a concentration of 1 µM (P < 0.05) and in CIEB at a concentration of 0.39 µM (P < 0.05). FB₁ had the greatest effect on lysosomal activity in REC starting at a concentration of 3.13 µM (P < 0.05) and in CIEB at a concentration of 6.25 µM (P < 0.05). Taken together, DON and FB₁ had a toxic effect on bovine rumen as well as calf intestinal epithelial cells.