The main objective of the present study was isolation and identification of soil streptomycetes, having antibacterial and antifungal activity against 8 selected phytopathogenic microorganisms, which attack crops in Slovakia. Out of 27 morphologically different streptomycete isolates, 14 of them demonstrated antimicrobial activity against at least two tested microorganisms in primary screening. Ethylacetate extract used for secondary screening showed different inhibitory pattern in comparison with primary screening. The tested isolates were mostly active against Gram-positive bacterium Clavibacter michiganensis subps. sepedonicus, the causal agent of bacterial canker of tomaten and against fungus Fusarium poae, pathogen of cereals, which can also infest stored grains. Only three isolates, namely VY59, VY87 and VY47 showed broad spectrum of inhibition activity and therefore were used for further identification. On the basis of various morphological (color of aerial and substrate mycelium, growth, production of pigments and melanin on ISP media), physiological (optimal pH, temperature, NaCl tolerance and C-utilization) and biochemical (ApiZym and ApiCoryne stripes) methods these strains belonged to the genus Streptomyces. In addition to the antimicrobial profile, the strains differed in API ZYM test results, which imply that the selected strains might produce different antimicrobial substances. Throught the comparative analysis of 16S rRNA gene, the most active isolates contained different nucleotide sequences for the 16S rRNA gene. Sequence similarity search by BLAST program revealed that they show sequence similarities to Streptomyces somaliensis (VY59), Streptomyces sp. (VY87) and Streptomyces albidoflavus (VY47). These three isolates with broader spectrum of antimicrobial activity can be used in the development of substances for agriculture purposes.