Abstract: Background and Objectives: Breast cancer is the second most common cancer in the world and the most frequent cancer among women. This study was conducted to investigate the anticancer activities of n-butanol extracts prepared from Streptomyces isolated from soils in Jordan against breast cancer MCF7 cells. Materials and Methods: After isolation and identification of Streptomyces isolates by conventional methods, n-butanol extracts were prepared from Streptomyces cultures. Hemolytic activity of extracts was determined. The cytotoxic effect of non-hemolytic extracts on normal MCF10A cells was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. The in vitro and in vivo selective cytotoxicity of extracts against breast cancer was estimated. The mechanism of action of extracts that exhibited in vivo cytotoxicity was determined. Isolates that gave in vivo cytotoxicity were identified by PCR amplification and sequencing. The IC50 values of extracts were determined by non-linear regression analysis. For tumor volume inhibition ratio, one-way ANOVA was applied for statistical evaluation of data and significant differences were considered significant at p<0.05. Results: The white aerial mycelia and the production of Rectus-Flexibilis (RF) sporophores as well as soluble pigments were the most common among Streptomyces isolates that screened from soil samples. The non-hemolytic n-butanol crude extracts of Streptomyces isolates (48 isolates) were screened for their cytotoxicity against normal breast MCF10A cells. Results indicated that out of the 23 non-toxic extracts on MCF10A cells, extracts of 9 isolates showed selective in vitro cytotoxicity against breast cancer MCF7 cells with IC50 values ranged from 0.68-1.64 mg mL–1. It was found that in vivo inhibition of breast cancer tumor in experimental animals was significantly increased, at α = 0.05, after treatment with extracts of 3 Streptomyces isolates (S7, S17 and S61). The DNA laddering (apoptosis feature) was observed in MCF7 cells treated with extracts of isolates S7 and S61. Analysis of 16S-23S rRNA gene sequence revealed that those 3 isolates have maximal identity to the genus Streptomyces. Conclusion: The result of the current study suggests that n-butanol extracts of 3 Streptomyces isolates have selective cytotoxicity against breast cancer MCF7 cells and 2 of the extracts induce apoptotic property in MCF7 cells.