Increasing multidrug resistance in Mycobacterium tuberculosis continues to diminish the number of effective drugs available for treatment of active tuberculosis. Although there are four new products (representing three new chemical classes) in clinical development, an active, robust pipeline of new chemical entities is critical to discovery of medicines to dramatically improve or shorten length of therapy via new mechanisms of action. In the absence of major pharmaceutical industry activity in tuberculosis drug development, the National Institute of Allergy and Infectious Diseases (NIAID) has supported the development of a high throughput screen for growth inhibitors of M. tuberculosis using a chemically diverse commercial library, a compound library available through the NIH Roadmap, Molecular Libraries Screening Center Network, and other compound sources. The rationale for these screens and suggested approaches for follow-up studies to identify compounds for advanced preclinical studies and as chemical probes of critical functions in M. tuberculosis, are discussed.