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According to studies, the microbiome may contribute to the emergence and spread of breast cancer. <i>E. coli</i> is one of the Enterobacteriaceae family recently found to be present as part of the breast tissue microbiota. In this study, we focused on the effect of <i>E. coli</i> secretome free of cells on MCF-7 metabolism. Liquid chromatography–mass spectrometry (LC-MS) metabolomics was used to study the <i>E. coli</i> secretome and its role in MCF-7 intra- and extracellular metabolites. A comparison was made between secretome-exposed cells and unexposed controls. Our analysis revealed significant alterations in 31 intracellular and 55 extracellular metabolites following secretome exposure. Several metabolic pathways, including lactate, aminoacyl-tRNA biosynthesis, purine metabolism, and energy metabolism, were found to be dysregulated upon <i>E. coli</i> secretome exposure. <i>E. coli</i> can alter the breast cancer cells’ metabolism through its secretome which disrupts key metabolic pathways of MCF-7 cells. These microbial metabolites from the secretome hold promise as biomarkers of drug resistance or innovative approaches for cancer treatment, either as standalone therapies or in combination with other medicines.