Tuberculosis (TB) remains a leading cause of death in the world today. The increase of drug-resistant TB is an increasing public health threat. There is therefore a critical need for discovering new, effective anti-TB drugs that target novel bacterial system to combat this disease. Mycobacterium tuberculosis, the etiological agent of TB, depends on the de novo biosynthesis of branched-chain amino acids (BCAA) for its replication in host cells. This biosynthesis pathway is present in bacteria, fungi, algae and plants but absent in humans. Thus, blocking this pathway may prove to be an effective anti-mycobacterial while representing a low risk of toxicity to humans. Acetohydroxyacid synthase (AHAS) is the first enzyme in the BCAA biosynthesis pathway. It is a promising target for the development of anti-mycobacterials. In this study, the herbicide family triazolopyrimidines (TP) were examined as inhibitors of M. tuberculosis AHAS (Mtb AHAS). The TP family member with the greatest potency is Florasulam (FS), with an MIC of 500 nM against the virulent Mtb strain H37Rv under normoxic conditions, and an MIC of 2.77 µM under hypoxic conditions, an important consideration to model the lung granuloma environment. Potency in these models, including effective intra-macrophage killing, a murine model of TB treatment was performed. In the mice study, FS treatment resulted in a significantly reduced the bacterial load of 1 log in the lung compared to vehicle treated mice. Thus, FS is a promising drug lead for anti-TB drug development and warrants further preclinical development and testing.