Benzothiazinones (BTZs) form a new class of potent antimycobacterial agents with a minimal inhibitory concentration of 1 ng/mL against Mycobacterium tuberculosis. Although the target of BTZs has been identified as decaprenylphosphoryl--D-ribose 2- oxidase (DprE1), their detailed mechanism of action remains obscure. This thesis provides new insights into BTZ’s mechanism of action demonstrating that these nitroaromatic compounds are activated in the bacterium by reduction of the essential nitro group to a nitroso derivative. This nitroso intermediate then specifically reacts in a near quantitative manner with an active-site cysteine of DprE1, thus providing a rationale for the extraordinary potency of BTZs. Further studies identify DprE1 as the nitroreductase responsible for BTZ activation and classify BTZs as suicide substrates. Mutant DprE1 enzymes from BTZ-resistant strains reduce BTZs to inert metabolites while avoiding covalent inactivation. This work thus provids a detailed picture of the mechanisms of BTZ sensitivity and resistance and represent an important step in the development of BTZs as anti-tuberculosis drugs.