Introduction Group B Streptococci (GBS) are a serious cause of morbidity and mortality in neonates. Infections can occur 1-7 days or up to 3 months after birth, leading to early and late onset disease, respectively (EOD/LOD). While intrapartum antibiotic prophylaxis protects against EOD, no prophylactic therapy exists against LOD with relatively high incidence for preterm neonates. Results We here present the characterization of Alpha-Like-Protein N-terminus (AlpN) reactive monoclonal antibodies (mAbs) derived from individual memory B cells isolated from subjects vaccinated in clinical trials with the Minervax GBS-AlpN vaccine. The mAbs showed reactivity and high binding affinity against one or several of the four AlpN antigens contained in the vaccine (AlphaC, Rib, Alp1 and Alp2/3). Epitope clustering highlighted the presence of 3-5 major binding clusters on each AlpN. Functionally, opsonophagocytic killing assay (OPKA) showed that single mAbs were efficient at mediating killing one of the Alp-serotypes. However, at least two mAbs targeting distinct epitopes were required to efficiently kill strains possessing one of the other three Alps. Finally, some of the mAbs with functional activity in the OPkA also possessed the ability to block GBS invasion of human cervical epithelial cells. Conclusion Antibodies against the AlpN vaccine antigens target functionally important epitopes. Such functional antibodies, if combined, can target all clinically relevant GBS strains. mAbs could thus become an important complement to a maternal vaccine for prevention of LOD, notably in preterm births.