The aim of this study was to assess the feasibility of using self-collected vaginal specimens for the quantitative real-time polymerase chain reaction (qPCR) assays of bacterial vaginosis (BV)-associated bacteria versus practitioner-collected swabs. A cross-sectional study included 190 pregnant women enrolled before 20 weeks’ gestation from September 2008 to November 2009. Self- and practitioner-collected swabs were taken during the same prenatal visit for each woman, qPCR assays performed for each, and the results compared. The quantification of the human albumin gene was used as an internal control to ensure sampling quality and accurate comparisons. The level of agreement of the qPCR assays for each microorganism was calculated with the Spearman product moment correlation coefficient and the kappa statistic. In all, 370 vaginal samples (185 self- and 185 practitioner-collected swabs) had a narrow range of values for the number of albumin gene copies and a significant correlation coefficient (Spearman’s rho = 0.532; p < 0.001). The agreement between both sampling methods was excellent (Spearman’s rho was 0.748 for Atopobium vaginae, 0.918 for Lactobacillus species, 0.940 for Gardnerella vaginalis; p < 0.001), especially for high concentrations of A. vaginae (≥10⁸ copies/mL; kappa value = 0.973; p < 0.001) and G. vaginalis (≥10⁹ copies/mL; kappa value = 0.903; p < 0.001). This study demonstrates the validity and reliability of self- versus practitioner-collected swabs for the molecular quantification of Lactobacillus species, G. vaginalis, and A. vaginae.