Leinamycin is a thiol dependent DNA alkylating agent which shows very potent activity against various cancer cell lines. This natural compound forms guanine adducts (N7) in DNA which are converted into a basic sites and simultaneously generates Reactive Oxygen Species (ROS), to produce DNA strand breaks in human cancer cells. In present study, eight different strains isolated from Iraqi soils were taxonomically assigned as Streptomyces.atroolivaceous. Remarkably the strain named as THS-44 was distinguished in productivity in comparison with other strains; the amount of leinamycin was 50.98 mg/l. In this study, we assessed the cytotoxic activity of leinamycin against RD and ANM3 cancer cell line in compare with REF cell line as a normal control. leinamycin revealed a potent activity against growth of both RD , and AMN3 cell lines with inhibition rate 93.56%, and 86.67%) respectively at 100mg/ml as a highest concentration. Moreover, leinamycin from HS-44 strain was mediate apoptotic effects on cancer cells without affecting on the normal cell line REF; a clear cut difference in the % of fragmented DNA was found between both RD, and AMN3 malignant cells, and REF normal cell line (89.16, and 83.1466, and 13.75; p< 0.001, and p< 0.001) respectively after exposure to 4.5μg/ml, leinamycin for 24hrs of incubation period.