Cervical shortening is a recognised risk factor for pre-term birth. The vaginal microbiome plays an essential role in pregnancy and in maternal and foetal outcomes. We studied the vaginal microbiome in 68 women with singleton gestation and a cervical length ≤25 mm and in 29 pregnant women with a cervix >25 mm in the second or early third trimester. Illumina protocol 16S Metagenomic Sequencing Library Preparation was used to detail amplified 16SrRNA gene. Statistical analyses were performed in R environment.Firmicuteswas the phylum most represented in all pregnant women. The mean relative abundance ofProteobacteriaandActinobacteriotawas higher in women with a short cervix. Bacterial abundance was higher in women with a normal length cervix compared to the group of women with a short cervix. Nonetheless, a significant enrichment in bacterial taxa poorly represented in vaginal microbiome was observed in the group of women with a short cervix.StaphylococcusandPseudomonas,taxa usually found in aerobic vaginitis, were more common in women with a short cervix compared with the control group, whileLactobacillus inersandBifidobacteriumwere associated with a normal cervical length.Lactobacillus jenseniiandGardenerella vaginaliswere associated with a short cervix.