Quercetin has been demonstrated to produce DNA damage in the presence of metal ions. In the present study, 7 natural and 5 semi‑synthetic glycosylated flavonoids were utilized to investigate the cupric ion (Cu2+)‑dependent DNA damage in vitro. The reaction mixture, containing single‑stranded DNA, different concentrations of flavonoids and cupric ion in the buffer, was incubated at three different temperatures. DNA damage was then assessed by gel electrophoresis followed by densitometric analysis. The reaction mixture with quercetin at 4, 20 and 54˚C induced DNA damage in a concentration‑ and temperature‑dependent manner. Furthermore, only the reaction at 54˚C resulted in DNA damage in flavonoids with glucosyl substitution of the hydroxyl group at the 3‑position on the C ring in quercetin. By contrast, loss of the hydroxyl group at the 3‑position on the C ring, or at the 3'‑ or 4'‑position on the B ring of quercetin, did not portray DNA damage formation at the investigated experimental temperatures. In addition, the experimental results suggested that the hydroxyl group at the 3‑position on the C ring produced the strongest capability to induce DNA damage in the presence of cupric ions. Furthermore, hydroxyl groups at the 3'‑ or 4'‑position on the B ring were only able to induce DNA damage at higher temperatures, and were less efficient in comparison with the hydroxyl group at the 3‑position on the C ring. Cupric ion chelating capacity was also assessed with spectroscopic analysis, and quercetin presented the largest chelating capacity among the tested flavonoids. Hydroxyl radical formation was assessed with a luminol reaction, and quercetin presented faster consumption of luminol. These results suggest that the 3‑position hydroxyl group of the C ring is required to induce DNA damage at low temperatures. Furthermore, the results of the present study also indicated that the presence of cupric ions will decrease the activity of the glycosylated quercetins, in terms of their ability to induce DNA damage.