This study aims to investigate the relation between vaginal microbiota and exposition to intra-amniotic inflammation (IAI). We conducted a prospective cohort study in women with preterm labor <34 weeks who had undergone amniocentesis to rule out IAI. Vaginal samples were collected after amniocentesis. Women with IAI included those with positive amniotic fluid (AF) for a microorganism identified by specific culture media and Sanger sequencing 16S ribosomal RNA gene and/or high AF interleukin (IL)-6 levels. Vaginal microbiota was characterized by 16S ribosomal RNA gene amplicon sequencing. Specific quantitative PCR targeted to Lactobacillus spp. was also performed. Regression models were used to evaluate associations between vaginal microbiota and exposition to IAI. Concerning our results, 64 women were included. We observed an inverse association between AF IL-6 levels and load of Lactobacillus spp. Depletion in Lactobacillus spp. load was significantly associated with an early gestational age at delivery and a short latency to delivery. Microbial-diversity was found to be a risk factor for the subsequent occurrence of clinical chorioamnionitis. To the contrary, higher Lactobacillus spp. load had a protective role. In conclusion, the study identifies reduced bacterial load of Lactobacillus spp. in women exposed to IAI and found microbial-diversity and Lactobacillus spp. depletion to be associated with a worse perinatal outcome.