Microbial dysbiosis in inflammatory bowel disease (IBD) reduces intestinal butyrate levels, compromising epithelial barrier integrity and enabling overgrowth of opportunistic pathogens such as Candida albicans. Here, we present a human immunocompetent colitis-on-chip model (CooC) that mimics key features of inflamed gut mucosa, including DSS-induced epithelial damage and C. albicans tissue invasion. Using this model, we uncover dual protective roles of microbiota-derived butyrate: (i) stabilization of epithelial adherens junctions and promotion of epithelial renewal, thereby restricting fungal invasion; and (ii) modulation of macrophage function to enhance antifungal activity while attenuating inflammasome-mediated inflammation. Butyrate pretreatment preserves barrier function, limits fungal translocation, and promotes macrophage viability through the inhibition of histone deacetylase (HDAC) and the suppression of NLRP3 inflammasome activation. These findings position butyrate as a key metabolite in orchestrating epithelial-immune defense against fungal exacerbation in colitis, supporting its therapeutic and preventive potential in restoring mucosal resilience in IBD.