Human milk oligosaccharides (HMOs) possess antimicrobial activity against a number of bacterial pathogens. HMOs prevent infection by serving as decoy receptors that competitively bind pathogens thus preventing pathogen attachment to host epithelial cell receptors. In a second mechanistic pathway, we recently demonstrated that heterogenous HMO extracts exert antimicrobial action against Group B Streptococcus by increasing cellular permeability. As human milk contains ca. 200 unique glycans however, our understanding of which pharmacophores are most important to HMO antimicrobial activity remains immature. In the present study, we describe the first evaluation of the antimicrobial and antibiofilm activities of five structurally defined, ubiquitous sialylated HMOs against Group B Streptococcus.