Fructooligosaccharides (FOS) are bioactive oligosaccharide fructans with beneficial health effects. Anti-hydroxyl radical activity is an important factor in the description of antioxidant capacity of any natural product. The aim of this study was to estimate in vitro anti-hydroxyl radical activity of the FOS 1-kestose and nystose by electron paramagnetic resonance spectroscopy (EPR) and fluorescence spectroscopy (FS) followed by a theoretical approach based on quantum chemistry calculations. A significant anti-hydroxyl radical potential of both compounds was observed (72% and 78% by EPR and 69% and 74% by FS, respectively), indicating the nystose to be a more active natural product. In addition, the computational results have confirmed that nystose follows the same pattern previously shown for 1-kestose, that is, that carbohydrates can react with hydroxyl radical. It is well known that FOS belong to cardioprotective nutraceuticals, so the study may be of some interest to research in heart disease.