Hygromycin B is an aminoglycoside antibiotic that kills bacteria, fungi and higher eukaryotic cells by inhibiting protein synthesis. It is biosynthesized in this research by Streptomyces sp. AZ151, isolated from soil sample in Assuit governorate and chosen among 194 actinomycete isolates. Morphological, physiological and biochemical culture characteristics as well as 16S rRNA nucleotide analysis confirmed strain AZ151 as Streptomyces crystallinus. An optimum environmental and nutritional condition in culture medium of this strain showed strong antibacterial and antifungal activities. Using organic solvent extraction, silica gel column chromatography and TLC, a high active metabolite, Hygromycin-B, was separated and identified as a suggested empirical formula of C15H30N2O10 and confirmed a biological efficiency.