Background: This study aimed to investigate the specific vaginal microbiome in the differential diagnosis of endometriosis/adenomyosis (EM/AM)-associated chronic pelvic pain (CPP) from other types of CPP, and to explore the role of the vaginal microbiome in the mechanism of EM/AM-associated CPP. Methods: We recruited 37 women with EM/AM-associated CPP, 25 women with chronic pelvic pain syndrome (CPPS) without EM/AM, and 66 women without CPPS into our study. All of the participants were free from human papillomavirus (HPV) infection. Sequencing of barcoded 16S rRNA gene fragments (V4) was used to determine the vaginal microbiome composition on the Illumina HiSeq2500 System.Taxonomic and functional bioinformatics analyses were performed using t-test, linear discriminant analysis effect size (LEfSe), MetaStat, and PICRUSt algorithms.Results: At the species level, EM/AM-associated CPP was found to be associated with a predominance of Clostridium butyricum, Clostridium disporicum, Alloscardovia omnicolens, and Veillonella montpellierensis, and a concomitant paucity of Lactobacillus jensenii, Lactobacillus reuteri, and Lactobacillus iners. When the relative abundance of Clostridium disporicum was over 0.001105% and that of Lactobacillus reuteri was under 0.1911349%, the differential diagnostic sensitivity and specificity were 81.08% and 52.0%, respectively.When serum CA125 was combined, the sensitivity increased to 89.19%, but the specificity remained at 52.0%. The PICRUSt results identified 7 differentially regulated pathways within the 3 groups that may be of relevance.Conclusions: Compared to that of CPPS patients without EM/AM and women without CPPS, the vaginal microbiome of patients with EM/AM-associated CPP shows significantly higher alpha (phylogenetic) diversity, as well as higher counts of Clostridium butyricum, Clostridium disporicum, Alloscardovia omnicolens, and Veillonella montpellierensis. These differences in the vaginal microbiome may interfere with local functional pathways, which could provide a direction for innovative metabolite-specific targeted treatment. The combination of vaginal biomarkers and serum CA125 may provide an original method to differentiate EM/ AM-associated CPP.