Oxidative stress is a risk factor for lifestyle-related diseases, such as cancer. Investigations of the factors that increase or decrease oxidative stress contribute to disease prevention. In the present study, we focused on the 8-hydroxyguanine (8-OHGua) in saliva, as a new oxidative stress biomarker. The relationship between lifestyles and salivary 8-OHGua levels in 541 Japanese subjects was analyzed. The salivary 8-OHGua levels were significantly elevated in older persons, as well as those who smoke, have hypertension, or excess visceral fat. By contrast, statistically significant lower levels of 8-OHGua were observed in persons who moderately exercised or recently drank green tea or coffee. The direct collection of saliva, without any special collecting device, was suitable for the 8-OHGua analysis. The present results suggest that oxidative stress can be measured in a non-invasive manner with easily collectable saliva, and the salivary 8-OHGua may be a useful biomarker for lifestyle-related disease prevention.