• The hypothesis was tested that flavonoids may scavenge singlet oxygen (¹O₂) in mesophyll cells of Phillyrea latifolia exposed to excess-light stress. • In cross-sections taken from leaves developed at 10% (shade) or 100% (sun) solar irradiance, we evaluated the excess photosynthetically active radiation (PAR)-induced accumulation of ¹O₂ in mesophyll cells by imaging the fluorescence quenching of the specific ¹O₂ probe N-[2-(diethylamino)ethyl]-N-[(2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-3-yl)methyl]-5-(dimethylamino)-1-naphthalenesulfonamide (DanePy). The intracellular location of flavonoids was also analyzed using three-dimensional deconvolution microscopy. • Photo-induced quenching of DanePy fluorescence was markedly greater in the mesophyll of shade leaves than in that of sun leaves, the former showing a negligible accumulation of mesophyll flavonoids. The photo-induced generation of ¹O₂ was inversely related to the content of flavonoids in the mesophyll cells of sun leaves. Flavonoids were located in the chloroplasts, and were likely associated with the chloroplast envelope. • Here we provide relevant evidence for the potential scavenger activity of chloroplast-located flavonoids against ¹O₂ and new insights into the photo-protective role of flavonoids in higher plants.