Abstract Objectives Elevated concentrations of numerous molecules have been reported in the peritoneal cavity of women with endometriosis. Until now, no factor proved sufficiently specific to endometriosis. We aimed to investigate several biomarkers in endometriosis and report their association with the menstrual cycle in a large sample size study. Methods Patients of reproductive age undergoing laparoscopic procedures for benign pathology in the Department of Obstetrics and Gynaecology, University of Bern between 2007 and 2018 were included. Exclusion criteria were the use of hormonal treatment in the three months prior to surgery, patients suffering from other inflammatory diseases, pregnancy, malignancy and surgery performed in an emergency. The concentrations of 13 different biomarkers in the peritoneal fluid (PF) were compared between patients with and without endometriosis both in the proliferative and the secretory cycle phase. Results Out of 1,256 patients in the database, 521 met the inclusion and exclusion criteria. Glycodelin (PP14) and Midkine concentrations were significantly higher in patients with endometriosis compared to controls irrespective of the cycle phase in which the PF was collected. Interleukin-8 (IL-8), regulated on activation normal T cell expressed and secreted (RANTES) and osteoprotegerin (OPG) concentrations were higher in patients with endometriosis only in the proliferative cycle phase. Monocyte chemoattractant protein 1, CCL2 (MCP-1) and Defensin concentrations were higher in patients with endometriosis only in the secretory cycle phase. Conclusions Certain pathophysiological processes may take place only in the one cycle phase leading to a temporary increase of specific PF biomarkers. Correlation with clinical outcomes is mandatory to establish their potential as prognostic or therapeutic tools in endometriosis.