Anti-cancer agents can be found in a variety of natural products because of their chemical and biological activity. Free radicals are molecules with high instability which induced initiation and progression of tumor formation. In contrast, the compositions of antioxidant material act as defense activator against free radical formation. In the present investigation, we prepared six different methanolic Fractions viz Fractions 1, Fractions 2. Fractions 3, Fractions 4, Fractions5 and Fractions 6 from the methanolic extract of Acacia catechu stem bark. Then we check the in vitro antioxidant activity of different extracted fractions compared with standard ascorbic acid. Based on the high potent antioxidant capacity Fraction 3 were further evaluated the antiproliferative effect on MCF-7 breast cancer cell line. However, MTT assay revealed that, methanolic extracted Fractions 3 had potent cytotoxic effects on MCF-7 cells in the IC50 value at 49.86μg/mL. The study suggested that, the isolated Fraction 3 form the stem bark of Acacia catechu act as preventive agent for free radical formation and also cytotoxicity effect on MCF- 7 cell line under laboratory condition.