AbstractPolycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds derived mostly from the incomplete combustion of fossil fuels and biomass. Human skin can absorb PAHs and the uptake increases with their molar mass and lipophilicity. Benzopyrene is high molecular weight PAH frequently appearing in ambient pollution. It exists in two isomeric forms: benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP), which exhibit different biological activity. Although certain properties of benzopyrenes suggested photoreactivity of the compounds, no direct measurements were previously conducted to characterize their photochemical activity. In this study, quantum yield and action spectra of singlet oxygen photogeneration by BaP and BeP were measured by time‐resolved near‐infrared phosphorescence, and the ability of both compounds to photogenerate superoxide anion was assessed by electron paramagnetic resonance (EPR) spin‐trapping. The measurements revealed high efficiency of benzopyrenes to photogenerate singlet oxygen and their ability to photogenerate superoxide anion. Using HaCaT cells as single‐layer skin model, we demonstrated concentration‐dependent and light‐dependent cytotoxicity of BaP and BeP. The compounds induced damage to the cell mitochondria and elevated the levels of intracellular reactive oxygen species.