Hydrogen water as a new therapeutic antioxidant has been widely used in living organisms under stress. In this study, we applied nanobubble (NB) technology to hydrogen water. The antioxidant capacity of hydrogen NB water was studied with respect to different reactive oxygen species (ROS) both in vitro and in vivo. Using a relatively weak reduced dye, APF, we showed that hydrogen NB water can effectively remove three cytotoxic ROS, (OH)-O-center dot, ClO-, and ONOO-, from water. Hydrogen NB water could also remove O-2(center dot-), which is a physiologically important ROS, from water. However, hydrogen water could not reduce other physiologically important ROS such as H2O2 and NO. At similar dissolved hydrogen concentrations, hydrogen NB water displayed higher antioxidant activity than hydrogen water without NB. Barley seed germination tests were used to study the antioxidant effect of hydrogen NB water on ROS generation in vivo. Our results showed that this decreased the physiological activity of barley seeds in their normal homeostatic state. Hydrogen NB water eliminated endogenous O-2(center dot-) in seeds and inhibited germination. The usage of hydrogen NB water should be individually considered according to the types of cells involved. Our results offer basic data concerning the application of hydrogen NB water in different fields.