Context: The most common cause of abnormal vaginal discharge in women of childbearing age is due to bacterial vaginosis (BV). This condition predisposes women to increased susceptibility to sexually transmitted diseases, including human immunodeficiency virus (HIV) infections and preterm birth. The diagnostic methods currently adopted in the evaluation of patient samples for BV are arguably Amsel criteria, and Nugent score that require microscopy and expert interpretation. The use of polymerase chain reaction (PCR) with group specific lactobacillus primers, in combination with Nugent criteria has not been widely used in the developing countries such as Nigeria. Objective: The aim of the present study was to examine the relationship between vaginal Lactobacilli using PCR with group specific lactobacillus primers and Nugent Score criteria for bacterial vaginosis. Study Design, Setting, Subjects, and Methods: We evaluated 241 vaginal samples from apparently healthy premenopausal women in Benin City for the presence of Lactobacilli with group specific primers in a polymerase chain reaction master cycler and bacterial vaginosis using the Nugent score criteria. Results: Of the 241 vaginal samples that were Gram stained for Nugent rating, 84 (34.8%) had Normal rating (0-3), 123 (51%) had Intermediate score (4-6), while 34 (14.2%) had Bacterial vaginosis, with Nugent score (7-10). There was a positive association between absence of lactobacilli PCR product and Nugent scores interpreted as BV (85.8% vs. 14.2%. ÷2 [2]= 4.12, P=0.05). Conclusion: The study has demonstrated a strong relationship between bacterial vaginosis and absence or depletion of vaginal lactobacilli using molecular techniques.