Background: Rectovaginal fistula is a major complication of surgery for deep endometriosis. Objective: To assess whether placement of a biological mesh (Permacol) between the vaginal and rectal sutures reduces the rate of rectovaginal fistula, in patients with deep rectovaginal endometriosis. Study Design: Retrospective, comparative study enrolling patients with vaginal infiltration > 3cm diameter and rectal involvement in two centers. They benefited from complete excision of rectovaginal endometriotic nodules, with or without a biological mesh placed between the vaginal and rectal sutures. Rectovaginal fistula rate was compared between the two groups. Results: 209 patients were enrolled: 42 patients underwent interposition of biological mesh (cases) and 167 did not (controls). 92% of cases and 86.2% of controls had rectal infiltration greater than 3cm in diameter. Cases underwent rectal disc excision more frequently (64.3% vs. 49.1%) and had a lower distance between the rectal stapled line and the anal verge (4.4+/-1.4 cm vs. 6+/-2.9cm). Rectovaginal fistulae occurred in 4 cases (9.5%) and 12 controls (7.2%). Logistic regression analyses revealed no difference in the rate of rectovaginal fistula following the use of mesh (adj OR 0.61, 95%CI 0.2-2.3). A distance < 7cm between the rectal stapled line and the anal verge was found to be an independent risk factor for the development of rectovaginal fistulae (adj OR 16.4, 95%CI 1.8-147). Conclusions: Placement of a biological mesh between the vagina and rectal sutures has no impact on the rate of postoperative rectovaginal fistula formation following excision of deep infiltrating rectovaginal endometriosis.