The gut microbiota plays a crucial role in protecting against enteric infection. However, the underlying mechanisms are largely unknown due to a lack of suitable experimental models. Whilst most gut commensals are anaerobic, intestinal epithelial cells require oxygen for survival. In addition, most intestinal cell lines do not produce mucus which provides a habitat for the microbiota. Here, we have developed a microaerobic, mucus-producing vertical diffusion chamber (VDC) model and determined the influence of Limosilactobacillus reuteri and Ruminococcus gnavus on enteropathogenic E. coli (EPEC) infection. Optimization of the culture medium enabled bacterial growth in the presence of mucus-producing T84/LS174T cells. While L. reuteri diminished EPEC growth and adhesion to T84/LS174T and mucus-deficient T84 epithelia, R. gnavus only demonstrated a protective effect in the presence of LS174T cells. Reduced EPEC adherence was not associated with altered type III secretion pore formation. In addition, co-culture with L. reuteri and R. gnavus dampened EPEC-induced interleukin-8 secretion. The microaerobic mucin-producing VDC system will facilitate investigations into the mechanisms underpinning colonization resistance and aid the development of microbiota-based anti-infection strategies.