The isoflavones daidzein and genistein are natural compounds which have anti-inflammatory and photoprotective activities, and may be effective in the repair of ultraviolet (UV)-induced photodamage. In this study, an alcoholic solution of aglycone isoflavones with a genistein:daidzein ratio of 1:4 [Rottapharm (RPH)-aglycone] was examined for its effects on the repair of DNA damage induced by a single dose of UVB irradiation (20 mJ/cm2). For this purpose, human skin cells were first UVB-irradiated and then treated with RPH-aglycone. Comet assay analysis was used to estimate the UVB-induced DNA damage at different time points after treatment by measuring the tail moment parameter. We found that treatment with 10 μmol/L RPH-aglycone solution resulted in a significantly reduced tail moment at 1 h after treatment, and 34–35% enhancement of damage repair at 4 h after treatment. These results suggest that isoflavone aglycones are protective against UVB-induced DNA damage.