Objective To identify a diagnostic microbial signature for endometriosis. Design Prospective cohort study Setting Nepean Hospital and UNSW Microbiome Research Centre, St George Hospital, Australia Population 64 age- and sex-matched subjects ( n=19 HC; n=24 N-ENDO and n=21 ENDO). All study participants, besides healthy controls, underwent laparoscopic surgical assessment for endometriosis, and histology was performed on excised lesions. Methods Oral, stool, and vaginal samples were self-collected at a single time point for healthy controls, and pre-operatively for patients undergoing laparoscopy. Samples underwent 16S rRNA amplicon sequencing, followed by bioinformatics analysis. Main Outcome Measures Compositional differences between cohorts as identified by diversity analyses, and differentially abundant microbial taxa, as identified by LEfSE analysis. Results The composition of the oral, stool, and vaginal microbiota is different between healthy controls and patients with and without endometriosis. Differentially abundant taxa are present within each cohort. Particularly , Fusobacterium was enriched in the oral samples from patients with moderate/severe endometriosis. Conclusions Distinct taxonomic and compositional differences were found between the microbiota in the mouth, gut and vagina of patients with and without endometriosis and healthy controls. Fusobacterium is noted as a key pathogen in periodontal disease, a common comorbidity in endometriosis. These findings support a role for the oral, vaginal, and stool microbiome in endometriosis, and present potential for microbial-based treatments and the design of a diagnostic swab.