The absence of a surface-lining mucus layer is a major pitfall for the Caco-2 epithelial model. However, this limitation can be alleviated by applying biosimilar mucus (BM) to the apical surface of the cell monolayer, thereby constructing a mucosa mimicking in vivo conditions. This study aims to elucidate the influence of BM as a barrier towards exogenic compounds such as permeation enhancers, and components of fed state simulated intestinal fluid (FeSSIF). Caco-2 cell monolayers surface-lined with BM were exposed to several compounds with distinct physicochemical properties, and the cell viability and permeability of the cell monolayer was compared to that of cell monolayers without BM and well-established mucus-secreting epithelial models (HT29-MTX-E12 cell monolayers and HT29-MTX-E12/Caco-2 cell co-culture monolayers). Exposure of BM-covered cells to constituents from FeSSIF revealed that it comprised a strong, hydrophilic barrier effect as 90% of BM-covered cells remained viable for >4 h, and the permeation rate of hydrophobic drugs was reduced. In contrast, the permeation rate of hydrophilic drugs was largely unaffected. Control monolayers displayed a loss of barrier function and <10% viable cells. The efficacy of fatty acid permeation enhancers were altered when investigated in BM-covered cells as compared to all the other studied epithelial models. Thus, Caco-2 cell monolayers surface-lined with BM constitute a valuable in vitro model that makes it possible to mimic intestinal fed state conditions when studying drug permeation.