Background: A normal vaginal microbiota may protect the vaginal mucosa from colonization by potentially pathogenic bacteria, including group B streptococci (GBS). The aim of this study was to investigate the association between colonization with GBS and the presence of specific vaginal microbiota isolated from vaginal swabs in the third trimester of pregnancy. Methods: A semiquantitative culture of 1860 vaginal swabs from consecutive pregnant women in their third trimester was analyzed. The dominant bacteria, including lactobacilli, were identified using MALDI-TOF mass spectrometry. An enrichment culture for GBS was performed on the swabs. GBS colonization correlated with the bacteria isolated at the same time. Results: Lactobacilluscrispatus was isolated in 27.5% of the cultures, followed by L. jensenii (13.9%), L. gasseri (12.6%), and L. iners (10.1%). The presence of lactobacilli as a group, and of L. crispatus, inversely correlated with GBS colonization (OR = 0.44 and OR = 0.5, respectively; both with p < 0.001). Other microorganisms, including Gardnerella vaginalis, mixed aerobic bacteria and yeasts, were not associated with GBS colonization. Conclusions: Lactobacilli, especially L. crispatus, may prevent GBS colonization in pregnancy. Maintaining a normal vaginal microbiota could be an effective method for the antibiotic-free prevention of invasive GBS infections in neonates.