A semi-quantitative multiplex PCR assay for the diagnosis of bacterial vaginosis (BV) was evaluated in a prospective study in a population of Dutch women with complaints of abnormal vaginal discharge. The PCR targets Gardnerella vaginalis, Atopobium vaginae, Megasphaera phylotype 1, Lactobacillus crispatus and Lactobacillus iners. Together with a short questionnaire, a vaginal swab for PCR and vaginal smear for microscopy were taken by their general practitioner or gynaecologist. Data from 151 women (median age 32) were available. Nugent Score (NS) was used to classify the samples and 83 samples were classified as normal (NS 0–3), 13 as intermediate (NS 4–6), and 55 as bacterial vaginosis (NS 7–10). In women with a NS of 7–10, PCR detected Gardnerella vaginalis, Atopobium vaginae and Megasphaera phylotype 1 in respectively, 96 %, 87 % and 60 %, whereas in women with a NS of 1–3 these species were detected in 27 %, 6 % and 2 % (P <0.001). A ratio of Lactobacillus crispatus over Lactobacillus iners of <1 (as calculated from the quantification cycle value (Cq)) was present in women with a NS of 7–10 in 66 % versus 33 % in women with a NS of 1–3 (P <0.001). The BV-PCR displayed a sensitivity of 92 % and specificity of 96 % with a positive predictive value of 94 % and a negative predictive value of 95 %. The Lactobacillus-index improved the correct classification of samples where only one of the other bacterial species was detected. Compared to the Nugent Score this multiplex qPCR offers a convenient tool for performing observer independent diagnosis of BV.