The aims of the present study were to isolate novel actinomycetes from the terrestrial soil and to evaluate their antibacterial potential. A total of 26 strains of actinomycetes were isolated and screened for antibacterial activity by the well diffusion method. Two of the isolates, VITAM-10 and VITAM-16, possessed a broad spectrum of antibacterial activity against the selected Gram positive and Gram negative ATCC bacterial strains. The isolate VITAM-16 produced a 22 mm zone of inhibition against Proteus vulgaris and VITAM-10 produced 17.6 mm zone of inhibition against Bacillus cereus. VITAM-16 exhibited potent antibacterial activity than VITAM-10. Hence, VITAM-16 was chosen for further studies. The isolate VITAM-16 was subjected to morphologic, cultural and biochemical characterizations and identified to be belonged to the genus Streptomyces. 16S rDNA nucleotide sequence of VITAM-16 showed 97 - 99 % sequences similarity with Streptomyces taxons. The ethyl acetate (EA) extract of VITAM-16 was analyzed by GC-MS, fumaric acid and propanoic acid were found to be the major compounds. The EA extract exhibited an MIC value from 0.06-0.90 mg/ml against tested bacterial pathogens. The results of the present study suggest that VITAM-16 can be explored further for identification of novel antibiotics. © 2017, Walailak University. All rights reserved.