The incidence of cancer especially breast cancer is increasing alarmingly world wide with a high percentage of death mainly in developing countries. Recently herbal medicines are coming to play a vital role in the reduction and prevention of cancer. The preliminary studies on a number of promising herbal drugs have anticancer potential. The current study investigates the in vitro cytotoxicity of decoction of a polyherbal formulation [Punarnava (Boerhaavia diffusa Linn.), Sunthi (Zingiber officinale Rosc.), Varuna (Crataeva nurvala Buch.Ham.)] in breast cell line (MCF-7). The selection of these drugs was based on Malayalam traditional treatment book ?Sarvarogachikitsanool? and also from the information that it was practiced by Ayurvedic physicians for breast cancer. The five different concentrations of test sample were used for in vitro cytotoxicity by MTT assay at 24 hours and 48 hours and the result exhibited cytotoxicity in breast cell line. The result were analyzed statistically and found that it was highly significant (p˂0.001). The IC 50 value was also calculated and the result obtained was 190?g/ml. The in vitro Lymphocyte viability assay was performed in normal lymphocytes. The result revealed that the test sample showed no significant toxicity in normal cells. Apoptosis was also checked by two staining methods (Acridine orange-Ethidium bromide stain and Hoesct stain), revealed that the test sample showed cytotoxicity by apoptosis. Thus the study showed that the polyherbal formulation possess cytotoxicity in breast cell line (MCF-7) and no toxicity in normal cells. Therefore, this might possess potentiality for the development of novel anticancer medicine