Garlic (Allium sativum) is a medicinal plant used as a food spice with numerous ethnopharmacological applications for treating illnesses. The study intended to determine whether the major phytocompoments found in active fraction of garlic extract have the ability to inhibit the growth of breast cancer MCF-7 cells in vitro. Different fractions of Allium sativum extract were subjected to MTT assay, and the most active Allium sativum ethyl acetate fraction (ASEAF) was selected for the examination of ROS estimation, apoptosis study, mitochondrial membrane potential (MMP), and cell cycle analysis by flow cytometer. MCF-7 cells treated with ASEAF exhibited apoptotic-like morphology; however, there was no substantial cytotoxicity towards the normal Vero cell line. The confirmation of apoptotic cell death and necrotic was achieved by the use of a double staining kit. Notably, ASEAF showed an increase in ROS level and a reduction in MMP. A cell-cycle investigation revealed that the ethyl acetate fraction arrested the cells in the S phase resulting in cellular apoptosis. In conclusion, this study supports the therapeutic application of ASEAF that could be effective against human breast cancer.