Synthesis and characterization of fluorescent benzo[α]phenoxazines viz., M-1B (10-chloro-6-methyl-7a,11a-dihydro-5H-benzo[α]phenoxazin-5-one), M-2B 6,10-dimethyl-7a,11a-dihydro-5H-benzo[α]phenoxazin-5-one), M-3B (6-methyl-7a,11a-dihydro-5H-benzo[α]phenoxazin-5-one) and benzo[α]phenthiazine, M-4B (6-methyl-5H-benzo[α]phenothiazin-5-one) were carried out. ¹H and ¹³C chemical shifts were assigned from the 2DgHSQCAD NMR experiments. Compound M-1B crystallizes in the orthorhombic space group P2₁2₁2₁, while M-2B and M-4B crystallize in the monoclinic space group P2₁/c. The crystal network of M-1B showed slipped π–π stacking and Cl⋯Cl interactions, while M-2B facilitated ladder like π–π stacked polymeric chains. The C⋯S contacts were observed in the crystal environment of M-4B. All these structures possess C–H⋯O interactions. Electronic structure and charge distribution in terms of molecular electrostatic potential and frontier orbital analyses based on the MO6-2X based density functional theory further showed that monomer and dimer structures are in keeping with the single crystal X-ray data and provide insights for the growth of the crystal network. Antiproliferative activity of M-1B–M-4B was determined from the MTT assay against a human breast adenocarcinoma cell line (MCF-7), human carcinoma cell line (HeLa) and normal skin cell line. All these compounds showed significant cytotoxic activity against MCF-7 and HeLa by inducing apoptosis and thus can be viewed as potential candidates for antitumor therapy. Compounds M-2B and M-4B were further found to be topoisomerase II inhibitors.