Extreme ecosystems can be a source of untapped micro-organisms able to produce novel bioactive compounds of industrial interest. Consequently, in this work, 32 Actinomycetes were isolated from 6 soil samples collected from Algerian Sahara in searching for untapped producers of novel antimicrobial compounds. All the isolates were further subjected to antimicrobial screening against pathogenic bacteria, yeast and fungi. The obtained results indicated that three of the isolates (named C, MS1 and 10) showed antimicrobial activities against most of the tested pathogenic micro-organisms. Therefore, these three promising isolates, previously identified as Streptomyces by morphological, biochemical and physiological methods, were selected for their subsequent identification by the whole cell matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. Thus, the isolates C, MS1 and 10 were identified as Streptomyces violaceoruber B263 UFL, Streptomyces albus B262 UFL, and Streptomyces badius B192 UFL, respectively. These results pointed out Actinomycetes from Sahara soils as potential sources of novel antimicrobial compounds. Also, MALDI-TOF MS showed to be a robust technique for bacteria identification.