To estimate the relationship between vaginal quantification of the main microorganisms related with bacterial vaginosis and the risk of preterm delivery among women with preterm labor. Molecular methods were used to prospectively quantify Lactobacillus species, Gardnerella vaginalis, Atopobium vaginae, and Mycoplasma hominis in vaginal fluid samples from women admitted for spontaneous preterm labor with intact membranes from July 2007 through July 2008. The primary outcome measure was the relationship between bacterial concentration at admission and preterm delivery, before 37 weeks of gestation. Sensitivity and specificity of molecular cutoff values and 95% confidence intervals (CIs) were calculated using the University of British Columbia Bayesian Calculator type 2. Of the 90 women included, 36 delivered before 37 weeks of gestation (40%). Preterm delivery was not associated with the presence of Lactobacillus species, G vaginalis, A vaginae, or M hominis. In contrast, molecular quantification detected high concentrations of A vaginae (10(6)/mL or more: 25.0% in the preterm group and 9.3% in the term group, P=.04) and G vaginalis (10(7)/mL or more: 16.7% and 3.7%, P=.03) more often in women with preterm deliveries compared with term deliveries. Moreover, high vaginal concentrations of these two microorganisms together were associated with a significantly (P=.03) shorter interval between preterm labor and delivery (46 days, 95% CI 30-61) than were lower concentrations (85 days, 95% CI 75-95). The hazard ratio for a short preterm labor-to-delivery interval was three times higher for high vaginal fluid concentrations of A vaginae and G vaginalis than for lower concentrations (hazard ratio 3.3, 95% CI 1.1-9.5, P=.03). The risk of preterm delivery is significantly associated with high vaginal concentrations of A vaginae and G vaginalis in women with preterm labor. ClinicalTrials.gov, www.clinicaltrials.gov, NCT00484653. III.