Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes serious infections in humans, notably cystic fibrosis. P. aeruginosa faces various stresses such as oxidative stress either in the environment or within the host during infection. In the present study, the influence of oxidative stress on both Pseudomonas antibiotic susceptibility and host pathogenesis was characterized. Prior exposure to H2O2 significantly altered P. aeruginosa susceptibility to tested antibiotics; colistin, ciprofloxacin, tobramycin, and ceftazidime. The minimum inhibitory concentrations (MICs) of tested antibiotics either increased or decreased following H2O2 exposure. Importantly, RT-qPCR revealed that expression of quorum sensing genes, that regulate virulence factors production in P. aeruginosa, was significantly higher in unstressed relative to H2O2-stressed cells. The impact of P. aeruginosa exposure to oxidative stress by H2O2 on bacterial pathogenesis was investigated using in vivo mice infection model. Interestingly, exposure to oxidative stress markedly reduces P. aeruginosa pathogenesis in mice. Unstressed P. aeruginosa was able to kill more mice as compared to H2O2-stressed bacteria. In addition, body weight of mice infected with unstressed P. aeruginosa was lower than that of mice inoculated with stressed bacteria. Isolated organs (spleen, liver, and kidney) from mice infected with unstressed bacteria exhibited increased weight as well as bacterial load in comparison with mice infected with stressed bacteria. In summary, current data highlight the impact of oxidative stress on P. aeruginosa antibiotic susceptibility as well as host pathogenesis. These findings could be helpful in treatment of infections caused by this important pathogen.