Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the most serious human bacterial diseases in terms of prevalence and mortality. Billions of people are infected and millions die annually. A limited number of antibiotics can be used to treat TB, some of which, like isoniazid, are only effective against mycobacteria. Resistance to these drugs has evolved, giving rise to multi-drug resistant TB, and recently to extensively drug resistant strains. There is a need for new anti-mycobacterial antibiotics. We have isolated a large (over 200 strains) collection of soil bacteria that secrete antibacterial compounds, assayed in competition assays in vitro on plates, against a variety of standard lab strains (Staphylococcus aureus, Pseudomonas aeruginosa, etc). Conditioned medium from these candidate antibiotic strains is used for preliminary characterization of the biochemical properties of the active compounds and starting material for chemical purification. From this collection, we have identified candidate strains whose compounds inhibit the growth of mycobacteria. Some candidates show a broad spectrum of activity, inhibiting the growth of two dozen common lab bacteria (Gram negative and positive), but one (Strain UD) inhibits only Bacillus megaterium, Mycobacterium smegmatis, and M phlei. Conditioned medium from UD cultures indicates that the active compound is relatively high molecular weight (over 50 KDa by size exclusion chromatography and over 10 KDa by dialysis), heat resistant (to 1210C), and inactivated by pronase but not proteinase K. Bacteria of strain UD are spore-forming, Gram positive aerobic/anaerobic rods, tentatively typed as Bacillus polymyxa. Preliminary results suggest that strain UD secretes a large heat-stable protease-sensitive compound capable of inhibiting the growth of mycobacterial test strains. Purification and further characterization of this compound will determine its potential for further testing as an antibiotic for treatment of mycobacterial disease. Other candidates from our collection with demonstrated activity against mycobacteria will also be investigated.