Objective Does the combined analysis of compounds in vaginal secretions plus vaginal microbiota composition improve the ability to predict risk for spontaneous preterm birth (SPTB) in women with a short cervix. Design Prospective observational study Setting Three hospitals in Brazil Samples Vaginal secretions from 568 women at 21-24 weeks gestation with cervical length measurement. Method Vaginal microbiome composition was determined by analysis of the V1-V3 region of the bacterial 16S ribosomal RNA gene. D- lactic acid and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in vaginal secretions were measured by ELISA. Main outcome measures SPTB in relation to cervical length and dominant vaginal bacteria. Results When Lactobacillus crispatus was the dominant bacterium the concentration of TIMP-1 was lower, and D-lactic acid was higher, than when either L. iners or Gardnerella vaginalis predominated (p < 0.001). By ROC analysis, a D-lactic acid level < 0.7 mM, and a TIMP-1 level > 1.5 ng/ml best predicted the absence of L. crispatus dominance. Vaginal levels of TIMP-1 were highest (p = 0.024) and D-lactic acid levels were lowest (p=0.032) in women with the shortest cervical length. The absence of L. crispatus dominance increased the sensitivity for predicting a SPTB from 22.4% to 81.8% or 78.3% in women with a cervical length <25mm or <30mm, respectively. Conclusions Low vaginal D-lactic acid and high TIMP-1 indicates the loss of L. crispatus dominance and increased risk for SPTB in women with a short cervix.