Both bacteria and fungus are important pathogens that have a significant impact on human health. It has always been crucial to investigate new medicinal compounds and other bioactive secondary metabolites from Streptomyces species for use in pharmaceutical and industrial applications. Streptomyces, a biologically potential melanin pigment, and its antagonistic activity against microbes were the focus of current research. To isolate actinomycete species that can produce the pigment melanin, eight soil samples from different regions of the Kafr El Sheikh Governorate were collected. Streptomyces isolates were characterized based on morphological, biochemical and cultural features and identified as Streptomyces aureofasciculus and given the name Streptomyces aureofasciculus M20. In this study, we used a secondary screening method to screen the antimicrobial activity of actinomycete metabolites against bacterial and fungal strains. Also the gamma irradiation at doses level (2.0 and 4.0 kGy) was used small doses as stress factor   to investigate   the effect of Streptomyces extract against bacterial and fungal strains. The SEM was used to exhibits distraction effect of both irradiated and non-irradiated extract, the results showed a significant   antibacterial and antifungal effect through morphological alteration on bacterial cells shape and size also morphological alteration on substrate and aerial hypha of   Aspergillus niger and significant effect on its sporangium. The chemical structure of the   extract was analyzed by using GC-MS which revealed the fractionation of extract as (Octadecenoic acid, Hexadecanoic acid Hexadecanoic  acid methyle ester, Octadecenoic acid(2-phenyl-1,3 Diooxolan-4-Y-)methyle ester Cis,9- Octadecenoic  acid methyle ester,1- Naphthalenoacetic acid-methyle ester,10- Octadecenoic  acid methyle ester and  -Octadecanoic  acid methyle ester) respectively and showing molecular formula (C18H34O2, C16H32O2, C17H34O2, C28H44O4, C19H36O2, C13H12O2, C19H36O2 and C19H38O2) respectively. Also the thin-layer chromatography, UV spectroscopy were used, the extract exhibits brown color, one spot, RF (0.8), wavelength 255 nm. The antitumor activity of the extract was studied against HCT-116 and MCF-7 cell line, the colon carcinoma cell line exhibits IC₅₀ = 15.1±0.9 µg/ml   and   the breast carcinoma cells exhibits IC₅₀= 24.7 ±1.2 µg/ml. So our study exhibited antimicrobial activity of the extract against both Gram negative Gram positive bacterial strains and fungal strain and significant effect after irradiation ad small dose level and also significant antitumor effect against both the colon and breast carcinoma cells in vitro.