This study aimed to evaluate the potential of oral probiotics to eradicate vaginal GBS colonization during the third trimester of pregnancy. We screened 1058 women for GBS colonization at 33–37 gestational weeks using a combination of vaginal-to-rectal swab and culture-based methods. Women who tested GBS positive were randomized to either the verum group, receiving a dietary probiotic supplement of four viable strains of Lactobacillus twice-daily for 14 days, or to the placebo group. Women underwent follow-up smears, whereat GBS colonization upon follow-up was considered the primary endpoint. We found that 215 women (20.3%) were positive for GBS upon screening, of which 82 (38.1%) were eligible for study inclusion; 41 (50%) of these were randomized to the verum and placebo groups each. After treatment, 21/33 (63.6%) members of the verum group, and 21/27 (77.8%) of the placebo group were still GBS positive (p = 0.24). Four (9.8%) women in the verum group and one (2.4%) in the placebo group experienced preterm birth (p = 0.20); smokers showed significantly higher rates of preterm birth (p = 0.03). Hence, the findings did not support the hypothesis that oral probiotics can eradicate GBS during pregnancy, although we observed a trend toward reduced GBS persistence after probiotic intake.