Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics (GPAs). The latter are canonical GPAs with broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridium difficile. Here, we investigated the three-dimensional structures and functional consequences for both molecules. Equilibrium binding studies showed tight binding by keratinimicin A, but not keratinicyclin B, to the peptidoglycan terminus. Using protein crystallography methods, we solved the X-ray crystal structures of both GPAs, which, in conjunction with DFT calculations, indicate that the inability of keratinicyclin B to bind the peptidoglycan is governed by steric factors. Keratinicyclin B, therefore, interferes with an alternative target to inhibit C. difficile growth, a conclusion confirmed by checkerboard analysis that revealed synergistic activity with vancomycin. Our results set the stage for identifying the molecular target of keratinicyclins and for exploring their therapeutic utility in combination with vancomycin.