Plants continue to play a critical role in the discovery of effective compounds, especially anticancer drugs. Abutilon pannosum has been historically used as a therapeutic plant in the medicinal system. In this study, an ethanolic crude extract was prepared from the dried powder of A. pannosum and subsequently fractionated to produce chloroform, butanol, and water fractions. The crude extract and fractions were tested for their cytotoxic action against various cancer cells using the MTT assay. Additionally, the effect of the most promising fraction on the cell cycle arrest and apoptosis induction was studied using flow cytometry and RT-PCR. Western blotting was employed to confirm the expression of apoptosis-related proteins. The chemical constituents of the most promising fraction were further analyzed by GC-MS. Among all the tested extracts, the A. pannosum chloroform fraction (APCF) exhibited the most potent activity against MCF-7 breast cancer cells, with an IC50 value of 50 µg/mL. The growth inhibition of the MCF-7 cells was found to be linked with cell cycle arrest at the G1 phase. Moreover, apoptosis was confirmed as a cell death mode using the FITC-annexin/PI assay, as well as the upregulation of proapoptotic genes, including Bax and caspase-7, and downregulation of the antiapoptotic Bcl-2 gene. The most abundant phytoconstituents revealed by the GC-MS analysis were palmitic acid (50.46%), quinic acid (11.84%), alpha-d-glucopyranoside (11.15%), parthenolide (9.65%), and phytol (6.65%). Our in vitro assessment indicates that A. pannosum could be a potential source of anticancer agents.