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The present study was attempted to determine the antimicrobial potential of actinomycetes from the Great Indian Thar Desert. A total of 100 different morphotypes based on phenotypic characterization were isolated from desert ecosystems located in the northwest of India and tested for their antimicrobial activity by the cross-streak method. Among the strains tested, 13 actinomycetes exhibiting strong antimicrobial activities against several test organisms, including multidrug resistant bacteria (MRSA) were chosen for a phylogenetic diversity study. The results of 16S rRNA gene sequencing showed their affiliation to actinobacterial genera: Actinomadura, Nocardia, Nonomuraea, Spirillispora, and Streptomyces. Three of these isolates were considered to be new members of the Streptomyces genus and another strain also seemed to be a new species of the genus Spirillispora. Among these strains, five were chosen to study the bioactive products using Q-Tof-MS because of their broad spectrum activity against the panel of test pathogens used. The results showed that they produce many known compounds and might produce few unknown compounds as well. This is the first such report on the selective isolation of actinomycetes from the Great Indian Thar desert, and their screening for antibacterial potential. This ecosystem has never before been explored to this extent.