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Abnormal vaginal colonisation can lead to adverse pregnancy outcomes such as preterm birth through intra-amniotic inflammation. Despite the concern, little is known about its risk factors and impact in pregnant women at high-risk for spontaneous preterm birth. Thus, we conducted this single-centre retrospective cohort study including 1381 consecutive women who were admitted to the high-risk pregnancy unit. The results of vaginal culture at admission were categorised according to the colonising organism: bacteria (Gram-negative or -positive) and genital mycoplasmas. Maternal baseline socioeconomic, and clinical characteristics, as well as pregnancy, delivery, and neonatal outcomes were compared according to the category. Maternal risk factors for Gram-negative colonisation included advanced maternal age, increased pre-pregnancy BMI, a greater number of past spontaneous abortions, earlier gestational age at admission, and IVF. Gram-positive colonisation was likewise associated with earlier gestational age at admission. Genital mycoplasmal colonisation was associated with a greater number of past induced abortions, a lower level of education completed, and a lower rate of multifetal pregnancy and IVF. The neonates from mothers with Gram-negative colonisation had a greater risk of NICU admission, proven early onset neonatal sepsis, and mortality. However, not Gram-positive bacteria or genital mycoplasma was directly associated with adverse pregnancy outcomes.