A natural polyphenol chlorogenic acid (CGA) is regarded as an antioxidant by preventing oxidative damage. Among the oxidative damages, DNA double-strand breaks (DSBs) are considered the most severe. To study the protective effect of CGA against DSBs, we measured the time-dependent increase of DSBs caused by photo-irradiation through a single molecule observation of DNA (166 kbp) using fluorescence microscopy. The results have been analyzed in relation to the two-step mechanism with single-strand breaks.