Tuberculosis remains the leading cause of death from an infectious disease, responsible for 1.3 million deaths annually. Here, we report the discovery of a first-in-class small molecule inhibitor targeting PurF, the first enzyme in the mycobacterial de novo purine biosynthesis pathway. Our lead candidate, JNJ-6640, exhibited nanomolar bactericidal activity in vitro. Using an array of comprehensive genetic and biochemical approaches, we confirmed JNJ-6640 was highly selective for mycobacterial PurF compared to the human homologue. Single-cell level timelapse microscopy demonstrated PurF inhibition leads to a downstream impact on DNA replication. Proof-of-concept studies using a long-acting injectable formulation demonstrated the compound's in vivo efficacy. Finally, we show inclusion of JNJ-6640 could play a crucial role in improving the current treatment regimen for drug-resistant TB. Taken together, we demonstrate that JNJ-6640 is a promising chemical lead and show that targeting purine metabolism is a novel strategy for TB drug development.