The present study was searching for actinomycetes isolates with antimicrobial activity from animals' dung. Twenty-two actinomycetes isolates were purified and nominated consistent with their culture features on starch nitrate agar medium. These purified isolates were surveyed for antimicrobial activity against different bacterial and fungal strains. Among all tested actinomycetes isolates, A12 isolate had the highest antimicrobial activity. The A12 isolate's morphological, physiological, and biochemical data all pointed to it being a member of the Streptomyces genus. The 16S rDNA gene sequence and phylogenetic connection of strain A12 revealed that it belongs to the Streptomyces hypolithicus HSM#10. The optimum conditions of growth and antimicrobial activity were submerged cultivation, temperature 30ᵒC, pH 7 for 7 days. Starch and potassium nitrate as best carbon and nitrogen source respectively and1% (w/v) NaCl. Ethyl acetate was used to extract antimicrobial metabolites from the A12 isolate, which were then examined using thin layer chromatography (TLC) which showed single spot. Ethyl acetate extract of the selected isolate was analyzed by UV, IR, HPLC and GC/MS. 1,2-Benzenedicarboxylic acid and diisooctyl ester was found to be the major component. MIC of purified extract was found to be 3.1, 3.1, 3.1, 6.3 and 25 mg/ml against B.cereus , S. aureus ATCC 29213, B. subtils , S.epedermadides and K.pneumonia ATCC53637 respectively. MBC of S. hypolithicus strain HSM#10 A12 extract against B.cereus , S.aureus ATCC 29213, B.subtils , S.epedermadides and K.pneumonia ATCC53637 were 6.3,3.1,12.5,6.3 and 25 mg/ml, respectively. S. hypolithicus HSM#10A12, was proved to have a broad-spectrum action against bacteria.