Two positional isomers of dibenzoxanthenes C₁ and C₂ were synthesized and characterized. The crystal structures of the compounds were solved by single-crystal X-ray diffraction. Binding the two compounds with CT-DNA (calf thymus DNA)/BSA (bovine serum albumin) was thoroughly investigated by UV-Vis and fluorescence spectroscopy. The compounds interact with DNA through an intercalative mode and show strong affinities with BSA. Their cytotoxicity upon irradiation was investigated in vitro using the MTT method. By comparing the two isomers, C₁ and C₂, compound C₂ showed greater enhanced therapeutic potential upon irradiation with visible light. Significant nuclear damage of HeLa cells was observed after a comet assay. These compounds inhibited cell growth in HeLa cells at the S phase. These compounds have been found to induce apoptosis in HeLa cells using AO/EB staining experiments and flow cytometry. The two compounds can enhance the intracellular reactive oxygen species and decrease the mitochondrial membrane potential. The compounds activated caspase-3, caspase-7, procaspase-7 and down-regulated the expression levels of the anti-apoptotic proteins Bcl-2 and Bcl-x, and up-regulated the expression levels of the pro-apoptotic proteins Bax and Bim. The compounds induced apoptosis of HeLa cells via a ROS-mediated mitochondrial dysfunction pathway.