Quercetin and other flavonoids have been reported to exhibit both antioxidant and pro-oxidant properties. Most studies about the pro-oxidative ability were conducted in the presence of metal ions, and the essential functional moiety of quercetin responsible for the pro-oxidative effect is still unclear. In this study, we evaluated the pro-oxidative abilities in the absence of metal ions of two quercetin derivatives, i.e., quercetin-3'-O-beta-D-glucoside (1) and quercetin-3-O-beta-D-glucoside (2), by assessing DNA cleavage and HO(*)-radical production. The binding mode between these compounds and DNA was studied by fluorescence and viscometric titrations. The results showed that 1 can efficiently induce oxidative damage to plasmid DNA, while 2 shows poor activity. Both 1 and 2 bind to DNA via groove-binding. These results proved that the alpha-hydroxy-beta-oxo-alpha,beta-enone moiety contributes to the pro-oxidative activity of quercetin.