Pseudomonas aeruginosa is a human pathogen causing mild skin to life-threatening bloodstream infections. Antibiotic treatment of P. aeruginosa is uneasy because the bacterium possesses intrinsic resistance mechanisms to various antibiotics and can acquire resistance to nearly all available antibiotics. It was reported that some antibiotics can induce oxidative stress that contributes to cellular death, but bacterial cells can detoxify oxidative stress by the oxidative stress-scavenging systems. The relative amount of antibiotic-induced oxidative stress to oxidative stress-scavenging systems may determine the roles of antibiotic-induced oxidative stress in cellular death. Glutathione is one of the oxidative-scavenging systems and is synthesized by glutamyl-cysteine synthetase encoded by gshA and glutathione synthetase encoded by gshB. This study aims to determine the roles of glutathione in oxidative stress and antibiotic susceptibility in P. aeruginosa. Glutathione-encoding genes were knocked out in P. aeruginosa PAO1, and the mutant strains (gshA::Gm, gshB::Gm, and gshA::Gm/gshB::Tc) were used to determine susceptibility to hydrogen peroxide, superoxide-producing paraquat, and antibiotics. The mutant strains were 2- to eightfold more susceptible to hydrogen peroxide and superoxide and 2- to fourfold more susceptible to all tested antibiotics than their parental strain. The susceptibility of hydrogen peroxide, superoxide, and antibiotics was genetically complemented in P. aeruginosa PAO1. Overall results indicate that glutathione is crucial in detoxifying oxidative stress induced internally and by antibiotics in P. aeruginosa. This finding suggests that glutathione is one of the oxidative stress-scavenging systems and one of the intrinsic resistance mechanisms to antibiotics and, thus, a potential drug target for P. aeruginosa.