Frequent occurrence of the mycotoxins deoxynivalenol (DON), enniatin B (ENB), beauvericin (BEA), ochratoxin A (OTA) and citrinin (CIT) in ruminant feed and feedstuff can be a potential threat to feed safety and ruminant health. Inadequate biodegradation of mycotoxins by rumen microflora following ingestion of mycotoxin-contaminated feeds by animals can lead to their circulatory transport to tissues such as mammary gland. Individual effects of these mycotoxins on barrier and innate immune functions of the bovine mammary epithelium were first investigated using a bovine mammary epithelial cell line (MAC-T) by measuring endpoints including cell viability, transepithelial electrical resistance (TEER), paracellular flux of FITC-40 kDa dextran and the expression of genes coding the tight junction (TJ) proteins zonula occludens-1, occludin and claudin 3, pattern recognition receptor toll-like receptor 4 (TLR4) and selected cytokines interleukin-6, tumor necrosis factor-α and transforming growth factor-β. The results indicated that all tested mycotoxins significantly decreased cell viability in a concentration-dependent manner. DON was more potent than BEA and ENB, whereas OTA was more potent than CIT. Tested mycotoxins differentially modulated TEER and FITC-dextran flux as well as mRNA expression of selected TJ proteins, pro- and anti-inflammatory cytokines and TLR4. Efficacy assessment of three yeast cell wall-based mycotoxin adsorbents namely, yeast cell wall, yeast cell wall extract and postbiotic yeast cell wall-based blend to mitigate the toxicity of DON, BEA, OTA and CIT was then carried out using an in vitro approach combining chemical-based assay and cell-based bioassay following the incubation conditions simulating the temperature and pH occurring in the gastrointestinal tract of dairy cattle. Adsorbent efficacy assessment indicated that Hill and Freundlich adsorption isotherm models were well-fitted into the LC-MS data; all tested adsorbents contributed to sequestering the tested mycotoxins to varying degrees and mitigating adverse effects of mycotoxins on MAC-T cells. The postbiotic yeast cell wall-based blend appeared to be the most efficacious overall. These findings support the hypothesis that the tested mycotoxins can disrupt barrier and innate immune homeostasis of bovine mammary epithelial cells, and that yeast cell wall-based adsorbents possess the capacity to adsorb selected mycotoxins.