Background: Bacterial vaginosis (BV) is a condition with diverse etiology. 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. These two methods are still subjective. Objective: The objective of this study was to determine the organisms present in the vagina of 34 HIV negative Nigerian women diagnosed as having bacterial vaginosis by using molecular techniques. Methods: The vaginal samples were subjected to DNA extraction, and amplified with eubacterial primers via PCR. The PCR products were separated using denaturing gradient gel electrophoresis (DGGE). Bands were excised, re-amplified, purified and sequenced. Sequence identification was performed using the BLAST algorithm and Genbank data base. Results: Mycoplasma hominis (12/34; 35%) was the most common isolate and 9 (26%) contained one of two clones of an unusual Rainbow Trout intestinal bacterium, while unculturable Streptococcus sp, and other bacteria made up the remaining isolates. Conclusions: The findings indicate further diversity in the etiological agents associated with BV, and raise the question as to whether diagnosis and management of this condition needs to be re-evaluated in countries like Nigeria. There is some controversy over the clinical importance of BV, as it was once regarded as a disease caused by Gardnerella and presenting as an odourous discharge condition, but is now diagnosed without necessarily the presence of these organisms or signs. With the incidence of BV aligned to an increased risk of HIV in a country ravaged by this virus, the effective eradication of BV can only be achieved if appropriate therapies are delivered. Author(s): Kingsley C Anukam1,2,3,4, | Gregor Reid1,5