Objective: To investigate the distribution of virulence genes in group B Streptococcus (GBS) causing infection in the third trimester of pregnancy and its effects on vaginal microecology and maternal-neonatal outcomes. Methods: A total of 200 pregnant women who had GBS infection in the third trimester and were admitted to our hospital from December 2021 to December 2025 were selected as the infection group. Meanwhile, 200 GBS-negative pregnant women in the third trimester were selected as the control group. Polymerase chain reaction was used to analyze the distribution of virulence genes in GBS. Vaginal microecology and maternal-neonatal outcomes were compared between the two groups. Results: In the 200 GBS strains, the most prevalent virulence genes were hyl B (100.00%), cyl E (100.00%), and scp B (96.50%), which were detected in different serotypes. The detection rate of lmb was 66.50%, and it was common in serotypes II, VI, and IX. The distribution of bac and bca was limited, with detection rates of 4.50% and 10.50%, respectively. The proportions of vaginal microecological imbalance, abnormal vaginal pH, and grades III–IV vaginal cleanliness in the infection group were all higher than those in the control group (P<0.05). The rate of cesarean section was higher (P=0.002), and the rate of natural vaginal delivery was lower in the infection group than in the control group (P=0.001). The incidences of intrauterine infection, premature rupture of membranes, puerperal infection, neonatal asphyxia, and neonatal pneumonia were all higher in the infection group (P<0.05). Conclusion: Virulence genes in GBS causing infection in the third trimester of pregnancy mainly include hyl B, cyl E, and scp B. GBS infection can lead to vaginal microecological imbalance and increase the risk of adverse maternal-neonatal outcomes.