The consumption of generally regarded as safe emulsifiers has increased, and has been associated with an increased prevalence of inflammatory bowel and metabolic diseases, as well as an altered microbiome. The mucus barrier, which selectively controls the transport of particulates and microorganisms to the underlying epithelial layer, has been previously shown to be altered by dietary salts and lipids. However, the potential impact of emulsifiers on the protective mucus barrier, its permeability, and associated structural changes are not clear. In this study, we analyzed changes in the mucus barrier to both passively diffusing nanoparticles and actively swimming E. coli upon exposure to two emulsifiers, carboxymethylcellulose (CMC) and polysorbate 80 (Tween). When exposed to CMC, mucus pore size decreased, which resulted in significantly slower E. coli speed and particle diffusion rates through mucus. Tween exposure minimally impacted mucus microstructure and particle diffusion, but increased E. coli speed in mucus. Moreover, both emulsifiers appeared to alter mucus amount and thickness in rat intestinal tissue and mucus-producing cell cultures. These results indicate that acute exposure to emulsifiers impacts barrier and structural properties of intestinal mucus, modulating interactions between intestinal lumen contents, microbes, and underlying tissue, which may contribute to development of intestinal inflammation.