The effects of l-glycine (Gly) and l-glutamic acid (Glu) on oxidative damage induced by hydrogen peroxide (H2O2) in Pediococcus pentosaceus R1 were investigated. Gly and Glu significantly reduce the production of intracellular reactive oxygen species and the levels of malondialdehyde and carbonylated proteins and concomitantly increase ATP levels in P. pentosaceus R1 under H2O2-induced stress (P < 0.05). Transmission electron microscopy and atomic force microscopy of bacteria under H2O2-induced stress revealed that Gly and Glu suppress bacterial membrane deformation and cell damage. Gly exhibited stronger ferrous ion-chelating ability, whereas Glu has higher radical scavenging activities and reducing power (P < 0.05). The abilities of Gly and Glu to inhibit lipid peroxidation are comparable. Gly and Glu significantly enhance the activities of superoxide dismutase and glutathione peroxidase, respectively, and increase the total antioxidant capacity of bacteria (P < 0.05). These findings indicate that Gly and Glu alleviate H2O2-induced oxidative stress via direct antioxidant effects and increase the activities of bacterial antioxidant enzyme.