Two series of novel chalcone derivatives containing acridin-9-yl or acridin-4-yl moiety have been synthesized, structure elucidated and further evaluated for their growth inhibitory activity against human cancer cell lines. Among the 12 investigated molecules, (2 E )-3-(acridin-4-yl)-1-(3,4,5-trimethoxyphenyl)-prop-2-en-1-one ( 4e ) exerted the best cytotoxic activity against aggressive and invasive, triple-negative breast cancer cell line MDA-MB-231 and estrogen responsive MCF-7 cells with the IC 50 values of 8.4 and 7.7 μM respectively and was selected for further studies. Furthermore, flow cytometry analysis showed 4e -induced, cell cycle block in the G2/M phase with a concomitant increase in the number of cells with sub-G0/G1 content, which is considered as a marker of apoptosis. Thereafter, we evaluated that compound 4e induced cell death by mitochondria-mediated apoptosis associated with loss of mitochondrial membrane potential (MMP), dysregulation of Bax and Bcl-xl protein expression, cytochrome c release, caspase 7 activation, and PARP cleavage. In addition, upregulation of p53 and p21 has also been observed. In vitro DNA interaction studies demonstrated that 4e interacts with CT DNA by bimodal binding mode: intercalation and groove-binding. Chalcone 4e showed an affinity to bovine serum albumin that indicates that can be efficiently transported by serum proteins in the bloodstream. This evidence fully confirmed that compound 4e could be a potential candidate that deserves further development as an antitumor agent against breast cancer. Graphical abstract