There is a well-documented need to replenish the antibiotic pipeline with new agents to combat the rise of drug resistant bacteria. One strategy is to discover new chemical classes, immune to current resistance mechanisms, which inhibit essential metabolic enzymes. Bacterial drug targets that have a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have very high selectivity for the bacterial enzyme. Our group is focused upon exploiting the essential metabolic enzyme biotin protein ligase (BPL). The X-ray crystal structure of Staphylococcus aureus BPL shows adjacent binding sites for the ligands biotin and ATP, making it an ideal candidate for a fragment-based approach to drug discovery. Although the residues at the biotin-binding site are highly conserved, the nucleotide pocket shows a high degree of variability that can be exploited to create selective compounds towards BPLs from pathogens. Screens were performed to find compounds that can occupy the nucleotide pocket of S. aureus BPL using a combination of in situ click chemistry, in silico docking, surface plasmon resonance and X-ray crystallography. The synthesis of new biotin 1,2,3 triazole analogues using click chemistry yielded our most potent structure (K_i 90 nM) with >1100-fold selectivity for the S. aureus BPL over the human homologue. The molecular explanation for the selectivity was explored using mutagenesis studies and identified a key arginine residue in the BPL active site necessary for selective binding. Importantly, the biotin triazole inhibitors showed cytotoxicity against S. aureus, but not cultured mammalian cells. We have demonstrated, for the first time that BPL from the clinically important pathogen Staphylococcus aureus can be selectively inhibited. The biotin 1,2,3 triazole provides a novel pharmacophore for future medicinal chemistry programmes to develop this new antibiotic class.