This study presents a scalable Gut Microbiome-on-a-Chip (GMoC) that enables high-resolution imaging and visualization of microbial interactions within a physiologically relevant 3D gut environment. The GMoC features a reproducible 3D stratified gut epithelium derived from Caco-2 cells (μGut), which mimics key intestinal architecture and functions. By incorporating tumorigenic bacteria, such as enterotoxigenic Bacteroides fragilis (ETBF), the GMoC allows for the observation of pathogenic behaviors, leading to μGut disruption and pro-tumorigenic signaling. Pre-treatment with beneficial microbes like Lactobacillus spp. prevents ETBF-mediated gut pathogenesis by promoting colonization resistance through competition. The GMoC provides a valuable tool for studying the roles of gut microbes in pathogenesis and developing microbial-based therapies. The GMoC successfully recapitulates the dynamic 3D μGut-microbe interface, enabling the visualization of microbial growth, behavior, and interactions. The μGut, generated from Caco-2 cells under perfusion, self-organizes into a physiologically relevant 3D epithelium with key attributes of a physiological intestinal epithelium, including a brush border and tight junctions. The μGut exhibits spatial patterning of differentiated cells and mucin production, creating an environment conducive to microbial colonization. ETBF colonization leads to pathogenic behaviors, including mucosal disruption, morphological damage, and activation of signaling pathways such as E-cadherin cleavage, β-catenin nuclear translocation, pStat3 nuclear translocation, and NF-κB activation, which are linked to colorectal cancer. However, pre-treatment with Lactobacillus spp. significantly reduces ETBF colonization and pathogenesis, preserving the healthy state of the μGut through inter-microbial competition. The GMoC offers a scalable and reproducible platform for studying gut-microbe interactions, providing insights into microbial behaviors and their impact on gut health. It enables the visualization of microbial colonization, growth, and pathogenic behaviors, as well as the mechanisms underlying microbial interactions. The study highlights the potential of the GMoC in uncovering the roles of gut microbes in disease and in developing microbial-based therapies.This study presents a scalable Gut Microbiome-on-a-Chip (GMoC) that enables high-resolution imaging and visualization of microbial interactions within a physiologically relevant 3D gut environment. The GMoC features a reproducible 3D stratified gut epithelium derived from Caco-2 cells (μGut), which mimics key intestinal architecture and functions. By incorporating tumorigenic bacteria, such as enterotoxigenic Bacteroides fragilis (ETBF), the GMoC allows for the observation of pathogenic behaviors, leading to μGut disruption and pro-tumorigenic signaling. Pre-treatment with beneficial microbes like Lactobacillus spp. prevents ETBF-mediated gut pathogenesis by promoting colonization resistance through competition. The GMoC provides a valuable tool for studying the roles of gut microbes in pathogenesis and developing microbial-based therapies. The GMoC successfully recapitulates the dynamic 3D μGut-microbe interface, enabling the visualization of microbial growth, behavior, and interactions. The μGut, generated from Caco-2 cells under perfusion, self-organizes into a physiologically relevant 3D epithelium with key attributes of a physiological intestinal epithelium, including a brush border and tight junctions. The μGut exhibits spatial patterning of differentiated cells and mucin production, creating an environment conducive to microbial colonization. ETBF colonization leads to pathogenic behaviors, including mucosal disruption, morphological damage, and activation of signaling pathways such as E-cadherin cleavage, β-catenin nuclear translocation, pStat3 nuclear translocation, and NF-κB activation, which are linked to colorectal cancer. However, pre-treatment with Lactobacillus spp. significantly reduces ETBF colonization and pathogenesis, preserving the healthy state of the μGut through inter-microbial competition. The GMoC offers a scalable and reproducible platform for studying gut-microbe interactions, providing insights into microbial behaviors and their impact on gut health. It enables the visualization of microbial colonization, growth, and pathogenic behaviors, as well as the mechanisms underlying microbial interactions. The study highlights the potential of the GMoC in uncovering the roles of gut microbes in disease and in developing microbial-based therapies.