Three 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes have been synthesized and investigated by biological, spectroscopic, crystallographic and computational methods. The spectroscopic and structural properties of the dyes have been investigated with UV, steady-state fluorimetry and X-ray methods. The spectral features have been characterized with respect to DFT and TD-DFT calculations. The cytotoxic and photocytotoxic abilities of the synthesized BODIPYs have been investigated in vitro using colon cancer SW480 cells. The cytotoxic effect of 4,4-difluoro-8-[4-(methoxycarbonyl)-phenyl]-1,3,5,7-tetramethyl-2-formyl-3a,4a-diaza-4-bora-s-indacene (1) after its addition and incubation with the cells has been examined by confocal spectroscopy. The photocytotoxicity of compound 1 has been tested on the colon cancer SW480 cells under varying the light dose and concentration of the added dye. The ability of the synthesized compounds to photogenerate singlet oxygen and superoxide anion has been measured by time-resolved near-infrared phosphorescence and electron paramagnetic resonance (EPR) spin-trapping, respectively.