This study presents a novel Gut Microbiome-on-a-Chip (GMoC) that provides a scalable and physiologically relevant platform for studying gut-microbe interactions. The GMoC features a 3D stratified gut epithelium derived from Caco-2 cells, mimicking key intestinal architecture and functions. This chip enables high-resolution imaging of microbial growth, behavior, and interactions, facilitating the visualization of individual and collective impacts on the gut. The GMoC incorporates a reproducible 3D μGut, which self-structures under shear flow conditions, mimicking the dynamic gut environment. The chip's design allows for easy integration with high-magnification microscopy, enabling detailed observation of microbial communities and their interactions. The study demonstrates the GMoC's ability to recapitulate pathogenic behaviors of enterotoxigenic *Bacteroides fragilis* (ETBF) in the μGut, leading to μGut disruption and activation of pro-tumorigenic signaling pathways. In contrast, pre-treating the μGut with beneficial gut microbes, such as Lactobacillus spp., effectively prevents ETBF-mediated gut pathogenesis, preserving the healthy state of the μGut through competition-mediated colonization resistance. The GMoC offers a valuable tool for exploring the unknown roles of individual gut microbes and microbial consortia in gut health and disease, providing insights into microbe-induced pathogenesis and potential therapeutic interventions. The platform's scalability and imaging capabilities enhance its potential for high-throughput assays and the development of targeted microbe-based therapies.This study presents a novel Gut Microbiome-on-a-Chip (GMoC) that provides a scalable and physiologically relevant platform for studying gut-microbe interactions. The GMoC features a 3D stratified gut epithelium derived from Caco-2 cells, mimicking key intestinal architecture and functions. This chip enables high-resolution imaging of microbial growth, behavior, and interactions, facilitating the visualization of individual and collective impacts on the gut. The GMoC incorporates a reproducible 3D μGut, which self-structures under shear flow conditions, mimicking the dynamic gut environment. The chip's design allows for easy integration with high-magnification microscopy, enabling detailed observation of microbial communities and their interactions. The study demonstrates the GMoC's ability to recapitulate pathogenic behaviors of enterotoxigenic *Bacteroides fragilis* (ETBF) in the μGut, leading to μGut disruption and activation of pro-tumorigenic signaling pathways. In contrast, pre-treating the μGut with beneficial gut microbes, such as Lactobacillus spp., effectively prevents ETBF-mediated gut pathogenesis, preserving the healthy state of the μGut through competition-mediated colonization resistance. The GMoC offers a valuable tool for exploring the unknown roles of individual gut microbes and microbial consortia in gut health and disease, providing insights into microbe-induced pathogenesis and potential therapeutic interventions. The platform's scalability and imaging capabilities enhance its potential for high-throughput assays and the development of targeted microbe-based therapies.