Background: bacterial vaginosis is universally the commonest vaginal infection in reproductively active females. It causes major consequences as preterm labor, predisposing to sexually-transmitted infections and HIV infections. Although it is a public health concern, no one knows exactly its pathogenesis when some say that it is just disturbance in vaginal floral balance predisposing to clinical symptoms and signs, where the predominant Lactobacilli in vagina become replaced by other facultative and anaerobic bacteria. Objective: To evaluate different diagnostic tests, Amsel's criteria and PCR and their ability to diagnose bacterial vaginosis in comparison to the gold standard test, Nugent score in terms of sensitivity, specificity, NPV and PPV. Also test for prevalence of BV among included females. Methodology: screening was done for all females in presence or absence of Amsel's criteria. Wet mount along with Gram stained films were examined in Microbiology lab and Nugent score was calculated for every patient. Cervico-vaginal aspirate samples were collected for detection of G. vaginalis, Lactobacilli and Sialidase enzyme by multiplex PCR. Results: Using Nugent score patients were categorized into bacterial vaginosis (BV) group (32%), non-BV group (51%) and 17 % were in intermediate group. Sensitivity, specificity, PPV and NPV of whole Amsel's criteria (100, 80.4, 76.2 and 100 % respectively) were better than using any criterion alone. Using multiplex PCR, detection of G. vaginalis (100%) and sialidase gene (93.7%) were higher in BV group and Lactobacilli gene (100%) higher in non-BV group with statistically high significant difference. Multiplex PCR detection of G. vaginalis has sensitivity (100%), specificity (92.2%), PPV (88.9%) and NPV (100%) for diagnosis of BV in relation to the gold standard test, Nugent score. Conclusion: using Amsel's criteria as a whole is better than using individual criteria for diagnosing BV with highest sensitivity, specificity, NPV and PPV. Combined PCR detection of G. vaginalis and sialidase gene can predict occurrence of virulent BV infection. BV is associated with significant loss of protective Lactobacilli.