Endometriosis (EMS) is a chronic disease characterized by unclear etiology, influenced by various genetic and environmental factors, with no definitive biomarkers available for early screening. Recent studies suggest that the abdominal microbiome and its metabolites, shaped by the host's genetic composition and environmental exposures, play significant roles in the pathogenesis and regulation of EMS. This study aims to integrate microbiome and metabolomics analyses to identify bacteria and metabolites critical to the progression of EMS. Multi–omics characterization was performed on pelvic cavity washings (PCW) from 7 EMS patients and 8 matched controls using 16S rRNA gene sequencing and liquid chromatography-mass spectrometry (LC–MS). A validation cohort of 42 EMS patients and 42 controls was assessed for clinical measures. Metabolomics revealed 7 metabolites across 12 metabolic pathways, with three uniquely distinguished in the endometriosis group. Microbial diversity analysis identified 2471 operational taxonomic units (OTUs), showing significant compositional disparities between groups. Notably, Pseudomonas enrichment in PCW was associated with advanced disease progression, especially in stage III and IV EMS, characterized by extensive pelvic adhesions and larger lesion sizes. Our findings highlight Pseudomonas as a potential biomarker for EMS severity, advancing the understanding of its pathogenesis and offering promising implications for non-hormonal therapeutic strategies.

Comprehensive analyses show significant interactions between Pseudomonas and its metabolite guanylate in endometriosis patients. Increased Pseudomonas levels correlate with larger lesions and more severe pelvic adhesions, suggesting it may serve as a biomarker and potential driver of disease severity, informing new preventive strategies and non-hormonal treatments.