The bioprospecting potential of actinomycetes from rare Indian ecosystems has been explored with particular reference to antituberculosis activity. Seventy-two actinomycete isolates were recovered from soil samples collected from different rare ecosystems, namely Thar desert, coffee plantation, rubber forest and hilly area. 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 surface agar fermentation using yeast extract malt extract agar. When screening for antituberculosis activity using the phage luciferase reporter assay, extracts from 39 actinomycetes showed activity against one or more M. tuberculosis isolates while 27 isolates showed antagonistic activity against nonmycobacterial pathogens. Notably, 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 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 are the promising source for the isolation of antituberculosis compounds. The active pigment produced by the actinomycete strain D25 derived from desert soil will likely be a potential candidate for the development of antituberculosis antibiotics.Keywords: actinomycetes, rare ecosystems, antituberculosis, LRP test, Thar DesertTuberculosis (TB) caused by Mycobacterium tuberculosis is a highly prevalent infectious disease...... at the center of the article...... aureus MTCC 96 and M. tuberculosis H37Rv which indicated the production of bioactive compounds even in liquid culture. Similar results have been obtained with submerged cultures by other authors [35,36]. Strain D25 isolated from the soil of Thar Desert, Rajasthan, showed activity exclusively against gram-positive bacteria and M. tuberculosis H37RV, drug-susceptible, MDR and XDR clinical strains of M. tuberculosis. The yellow-orange bioactive extracellular pigment was found to be produced only on solid medium but not on liquid medium. The promising activity exhibited by strain D25 against 15 clinical isolates of M. tuberculosis showing different patterns of antibiotic resistance further strengthens its potential. The results of the present study highlighted that actinomycetes of Indian rare ecosystems deserve the potential for bioprospecting with particular reference to antituberculosis compounds.