The purpose of this study was to investigate the effects of antioxidant biofactor (AOB) on reactive oxygen species (ROS). Generation of superoxide radical (O₂^•−) and hydroxyl radical (^•OH) was determined using an electron spin resonance (ESR) spin-trapping method. AOB was added at different concentrations to these free radical generating systems. The generation of both O₂^•− and ^•OH was scavenged by the addition of AOB in a dose-dependent manner. These results indicate that AOB has strong antioxidant properties against these radicals. We further investigated the anti-oxidative effect of AOB on human gingival fibroblasts (HGFs). HGFs were treated for 3 h with α-MEM containing a combination of AOB and H₂O₂ (AOB + H₂O₂ group), containing H₂O₂ (H₂O₂ group), or containing AOB alone (AOB group). Non-stimulated HGFs were used as a control group. The number of surviving cells was in the order of the AOB group > control group > AOB + H₂O₂ group > H₂O₂ group. The level of expression of type I collagen mRNA and production of collagen were also in the order of the AOB group > control group > AOB + H₂O₂ group > H₂O₂ group. In conclusion, our results suggest that AOB may protect HGFs against oxidative stress by reducing stress-induced ROS.