Streptococcus agalactiae (Group B Streptococci, GBS) can cause severe neonatal sepsis. The recto-vaginal GBS screening of pregnant women and intrapartum antibiotic prophylaxis (IAP) to positive ones is one of the main preventive options. However, such a strategy has some limitations and there is a need for alternative approaches. Initially, the vaginal microbiota of 30 non-pregnant and 24 pregnant women, including the assessment of GBS colonization, was studied. Among the Lactobacillus isolates, 10 Lactobacillus salivarius strains were selected for further characterization. In vitro characterization revealed that L. salivarius CECT 9145 was the best candidate for GBS eradication. Its efficacy to eradicate GBS from the intestinal and vaginal tracts of pregnant women was evaluated in a pilot trial involving 57 healthy pregnant women. All the volunteers in the probiotic group (n = 25) were GBS-positive and consumed ~9 log10 cfu of L. salivarius CECT 9145 daily from week 26 to week 38. At the end of the trial (week 38), 72% and 68% of the women in this group were GBS-negative in the rectal and vaginal samples, respectively. L. salivarius CECT 9145 seems to be an efficient method to reduce the number of GBS-positive women during pregnancy, decreasing the number of women receiving IAP during delivery.