Inhibition of the abnormal proliferation of Escherichia coli is crucial for alleviating inflammatory bowel disease. Due to the complexity of the gut microbiota, specifically targeting and inhibiting particular microbes is a challenging task. One approach involves screening for probiotics that antagonize E. coli; however, this process often requires extensive in vitro experiments using a vast pool of probiotics. In this study, we employed a genome-scale metabolic prediction model to predict the interactions between 803 strains of microbes and E. coli. The results indicated that Bifidobacterium breve and Bifidobacterium animalis exhibited the most potent inhibitory effects. In vitro antibacterial assays and mouse experiments validated the inhibitory efficacy of B. breve CCFM1369. Additionally, B. breve CCFM1369 demonstrated the ability to inhibit key gene Ldha and HIF-2α and reduce the luminal lactate concentration, thereby diminishing the colonization capacity of E. coli. These findings support the potential application of B. breve in the alleviation of colitis.