The bioprospecting potential of actinomycetes from rare Indian ecosystems was explored with special reference to anti-tuberculosis activity. Seventy-two actinomycete isolates were recovered from soil samples collected from different rare ecosystems, namely Thar desert, coffee plantations, rubber forest and hill region. After dereplication of 18 actinomycetes based on their phenotypic characteristics, fifty-four actinomycete isolates were selected among which 43 isolates were streptomycetes and 11 were rare actinomycetes. Crude extracts of actinomycete isolates were produced by agar surface fermentation using yeast extract and malt extract agar. When screened for antituberculosis activity by the phage luciferase reporter assay, extracts of 39 actinomycetes showed activity against one or more M. tuberculosis isolates, while 27 isolates exhibited antagonistic activity against nonmycobacterial pathogens. In particular, crude extracts of sixteen actinomycete isolates inhibited all three M. tuberculosis isolates tested. Among these 16 isolates, strain D25, isolated from Thar Desert soil, produced a yellow-orange pigment only in solid culture, showing promising activity against 15 clinical isolates of M. tuberculosis and Gram-positive bacterial pathogens. The results of the present study revealed that actinomycetes from Indian rare ecosystems provide a promising source for the isolation of anti-tuberculosis compounds. The active pigment produced by the desert soil-derived actinomycete strain D25 will likely be a potential candidate for the development of anti-tuberculosis antibiotics. Keywords: actinomycetes, rare ecosystems, anti-tuberculosis drugs, LRP test, Thar Desert Tuberculosis (TB) caused by Mycobacterium tuberculosis is a very common infectious disease... middle of article...... aureus MTCC 96 and M. tuberculosis H37Rv which also indicated the production of bioactive compounds in the liquid culture. Similar results were obtained with submerged cultures by other authors [35,36]. Strain D25 isolated from Thar Desert soil, Rajasthan, showed activity exclusively against Gram-positive bacteria and M. tuberculosis H37RV, drug-susceptible clinical strains, MDR and XDR of M. tuberculosis. . The extracellular bioactive yellow-orange pigment was found to be produced only in solid media but not in liquid media. The promising activity exhibited by strain D25 against 15 M. tuberculosis clinical isolates with different antibiotic resistance profiles further strengthens its potential. The results of the present study demonstrated that actinomycetes from Indian rare ecosystems merit bioprospecting potential, with special reference to anti-tuberculosis compounds..