To analyze the effects of vaginal microecological imbalance and group B streptococcus (GBS) infection during the third trimester of pregnancy on maternal and infant outcomes. Methods: 318 pregnant women during the third trimester of pregnancy were selected as the study subjects from May 2016 to May 2018. The secretions 1/3 from the vaginal side wall and crissum of pregnant women were collected and conducted microbiological examination. The real time fluorescent PCR-probe method was used for GBS tested. The effects of vaginal microecological imbalance and GBS infection on maternal and infant outcomes were analyzed. Results: Among the 318 pregnant women, 21.1% women had microecological imbalance, and the GBS infection rate was 9.8%. The incidence of abnormal vaginal pH of women with vaginal microecological disorders or positive GBS infection was 61.2% or 67.7%, which was significant higher than that (23.5% or 27.5%) of women with normal microecological conditions or negative GBS infection (all P<0.05). The incidences of cesarean section, premature rupture of membranes, fetal distress, postpartum hemorrhage, and neonatal jaundice, asphyxia, and pneumonia of women with vaginal microecological disorders or positive GBS infection were significant higher than those of women with normal microecological conditions or negative GBS infection (all P<0.05). Conclusion: Vaginal microecological imbalance and GBS infection of women during the third trimester of pregnancy are closely related to adverse maternal and infant outcomes.