Coumarins have demonstrated a broad spectrum of pharmacological activities, including significant anticancer properties. Recently, a series of cyclocoumarol derivatives, a pyranocoumarin known for its anticoagulant and anti-inflammatory effects, have been synthesized and shown to inhibit cyclooxygenase-2 (COX-2) activity selectively. In this study, we evaluated the anticancer effects of these cyclocoumarol derivatives for the first time. We tested their antiproliferative effects on several human cancer cell lines, including MDAMB231, A549, MCF7, SF268, HCT116, HeLa, and Jurkat. The MTT assay revealed that the methylated cyclocoumarol derivative, 2-methoxy-2-methyl-(1-(p-tolyl))-3,4-dihydropyrano [3,2-c] chromen-5(2H)-one, 5b exhibited the most potent antiproliferative activity, particularly against MDAMB231 cells. Further investigations demonstrated that 5b induced apoptosis in MDAMB231 cells via the mitochondrial pathway, as indicated by increased Bax and decreased Bcl2 levels, along with caspase-3 activation and PARP cleavage. Additionally, compound 5b significantly inhibited the clonogenic potential of MCF7 cells and reduced the migration capacity of MDAMB231, SF268, and A549 cells. These findings suggest that 5b is a promising lead compound for developing new anticancer agents, with the potential for chemical optimization to enhance its efficacy.