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Abstract Background Endometriosis is a gynaecological condition characterised by immune cell infiltration and distinct inflammatory signatures found in the peritoneal cavity. In this study, we aim to characterise the immune microenvironment in samples isolated from the peritoneal cavity in patients with endometriosis. Methods We applied mass cytometry (CyTOF), a recently developed multiparameter single-cell technique, in order to characterise and quantify the immune cells found in peritoneal fluid and peripheral blood from endometriosis and control patients. Results Our results demonstrate the presence of more than 40 different distinct immune cell types within the peritoneal cavity. This suggests that there is a complex and highly heterogeneous inflammatory microenvironment underpinning the pathology of endometriosis. Stratification by clinical disease stages reveals a dynamic spectrum of cell signatures suggesting that adaptations in the inflammatory system occur due to the severity of the disease. Notably, among the inflammatory microenvironment in peritoneal fluid (PF), the presence of CD69+ T cell subsets is increased in endometriosis when compared to control patient samples. On these CD69+ cells, the expression of markers associated with T cell function are reduced in PF samples compared to blood. Comparisons between CD69+ and CD69− populations reveal distinct phenotypes across peritoneal T cell lineages. Taken together, our results suggest that both the innate and the adaptive immune system play roles in endometriosis. Conclusions This study provides a systematic characterisation of the specific immune environment in the peritoneal cavity and identifies cell immune signatures associated with endometriosis. Overall, our results provide novel insights into the specific cell phenotypes governing inflammation in patients with endometriosis. This prospective study offers a useful resource for understanding disease pathology and opportunities for identifying therapeutic targets.