Human milk oligosaccharides (HMOs) possess antimicrobial and antibiofilm activity against Group B Streptococcus (GBS). HMOs were screened for their ability to potentiate antibiotic activity. We observed that HMOs potentiate the function of aminoglycosides, lincosamides, macrolides, and tetracyclines on a strain specific basis but not β-lactams or glycopeptides that inhibit cell wall synthesis. These findings are notable as GBS has evolved high levels of resistance toward aminoglycosides, macrolides, and tetracyclines. Finally, HMOs potentiate the function of aminoglycosides against both Staphylococcus aureus and Acinetobacter baumannii. On the basis of these observations, we hypothesized that HMOs act by increasing membrane permeability. This hypothesis was evaluated using a bacterial membrane permeability assay which revealed that HMOs increase membrane permeability toward propidium iodide.