BACKGROUND: Endometriosis, a poorly studied gynecological condition, is characterized by the presence of ectopic endometrial lesions resulting in pelvic pain, inflammation, and infertility. These associated symptoms contribute to a significant burden, often exacerbated by delayed diagnosis. Current diagnostic methods involve invasive procedures, and existing treatments provide no cure. METHODS: Microbiome-metabolome signatures in stool samples from individuals with and without endometriosis were determined using unbiased metabolomics and 16S bacteria sequencing. Functional studies for selected microbiota-derived metabolites were conducted in vitro using patient-derived cells and in vivo by employing murine and human xenograft pre-clinical disease models. FINDINGS: We discovered a unique bacteria-derived metabolite signature intricately linked to endometriosis. The altered fecal metabolite profile exhibits a strong correlation with that observed in inflammatory bowel disease (IBD), revealing intriguing connections between these two conditions. Notably, we validated 4-hydroxyindole, a gut-bacteria-derived metabolite that is lower in stool samples of endometriosis. Extensive in vivo studies found that 4-hydroxyindole suppressed the initiation and progression of endometriosis-associated inflammation and hyperalgesia in heterologous mouse and in pre-clinical models of the disease. CONCLUSIONS: Our findings are the first to provide a distinct stool metabolite signature in women with endometriosis, which could serve as stool-based non-invasive diagnostics. Further, the gut-microbiota-derived 4-hydroxyindole poses as a therapeutic candidate for ameliorating endometriosis. FUNDING: This work was funded by the NIH/NICHD grants (R01HD102680, R01HD104813) and a Research Scholar Grant from the American Cancer Society to R.K.