Abstract: This research aims to isolate and identify some actinomycetes, that having antimicrobial substances against some pathogenic bacteria and fungi, from soil samples in Al-Khurmah governorate. Only one actinomycete culture KSA-20 exhibited to produce wide spectrum of antibacterial activities. The nucleotide sequence of the 16S rRNA gene of this strain evidenced an 95% similarity with Streptomyces griseoviridis. From the taxonomic features, the actinomycete isolate KSA-20 matches with Streptomyces griseoviridis in the morphological, physiological and biochemical characters. Thus, it given the suggested name Streptomyces griseoviridis KSA-20. The parameters controlling the biosynthetic process of antibacterial agent were fully investigated. The separation of the active ingredient and its purification were performed using both Thin Layer Chromatography (TLC) and Column Chromatography (CC) techniques. The minimum inhibition concentrations “MICs” of the purified antibiotic were also determined. The purified antibiotic suggestive of being belonging to griseoviridin antibiotic produced by Streptomyces griseoviridis KSA-20.