Background: The association of genital microorganism with infertility has been documented but no consensus exists. Understanding their prevalence amongst infertile clients may assist in facilitating better screening protocols. The objective of this study is to determine the prevalence of microorganisms routinely screened among women undergoing infertility evaluation at the University of Benin Teaching Hospital. Methods: A three year (January 2015 to December 2017) retrospective survey of all patients evaluated for infertility at the assisted reproduction unit of the hospital was undertaken. Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis, cytomegalovirus (CMV), hepatitis B (HBV), hepatitis C (HCV) virus and the human immunodeficiency virus (HIV) were microorganisms serologically assayed at the unit. We analyzed data containing patients' demography and results of serological assay of these microorganisms. Results: There were 576 patients (288 couples) who completed their microbiological evaluation during the study period. The mean age (years) of female partners was 38.2±5.7, while the mean age of the male partners was 42.7±6.1. The frequency of CMV positive assay for infertile couples was 129 (22.4%); C. trachomatis 125 (21.7%); M. hominis 92 (15.9%) and U. urealyticum 76 (13.2%). Overall, more women (50.7%) were seropositive compared to men (26%). HIV was positive in 10 patients (1.73%) with 60% being women. HBV was seropositive in 8 (1.4%) (women 62.5% and men 37.5%) while HCV was positive in only 2 (0.3%) patient. Majority (over 80%) of couples were serodiscordant with 20% (2) concordance rate for HIV and 12.5% (1) for hepatitis B. Conclusion: Despite a relatively high seroprevalence rate of the studied microorganisms, the documented uncertainty on their association with infertility or its treatment limits justification for incorporation of routine screening of microbiological organisms into standard protocols for evaluation of infertile couples. A robust study on the impact of genital microorganism on specific infertility variables with comparison to fertile controls is recommended.