Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). It is estimated that one third of the world’s population is infected with TB resulting in 1.7 million deaths each year. Further complicating the TB epidemic is the emergence of drug resistant strains, including a strain resistant to all current anti-TB drugs (TDR). Consequently, new antibiotic classes that are not subject to current resistant mechanisms are urgently required. One novel approach to anti-TB discovery is to identify small molecules that target the utilisation of biotin. Biotin is an important enzyme cofactor that is post-translationally attached to specific biotin-dependent enzymes by biotin protein ligase (BPL). BPL plays an essential metabolic role in M. tuberculosis and, therefore, represents a promising new anti-TB drug target. As humans also possess a BPL homologue, it is important to discover inhibitors that are selective for the bacterial pathogen, but not the human equivalent. Our team has designed, synthesised and tested chemical analogues of the adenylated reaction intermediate employed by BPLs as a route to new anti-TB agents. In vitro enzyme analysis revealed one analogue that was a potent BPL inhibitor with anti-TB activity. A second class, the biotin triazoles, showed excellent selectivity for the M. tuberculosis BPL but not the human equivalent, thereby providing a therapeutic window for selective BPL inhibitors. A library of biotin triazoles was synthesised and tested yielding a structure – activity relationship series. This data from this SAR series will now be used chemically optimise the next generation biotin triazoles with potential for combating drug resistant TB.