Purpose of investigation: The aim of this study was to investigate if vaginal preparation procedure affects the occurrence of oocyte pickup-associated pelvic inflammation (OPU-PI) and the reproductive outcome in an in vitro fertilization (IVF) program. Materials and Methods: The occurrence of OPU-PI and the reproductive outcome were compared between 956 infertile patients undergoing vaginal preparation with saline douching alone versus 1,216 infertile patients undergoing a combination of povidone iodine disinfection and subsequent saline douching in an IVF program. Results: OPU-PI occurred in four patients (0.042%) in the saline douching alone group, whereas there were no cases in the combination group (p = 0.016). There were no significant differences in the rate of fertilization, morphologically good embryo acquisition, clinical and ongoing pregnancy between the two groups (p > 0.23). Conclusions: This large cohort study demonstrated that a combination of vaginal povidone iodine disinfection and subsequent saline douching is more effective procedure than saline douching alone to prevent OPU-PI, without spoiling the oocyte quality.