The accumulation of reactive oxygen species (ROS) during biotic stress is either part of a hypersensitive response of the plant or induced directly by the pathogen. Antioxidants such as ascorbate and glutathione counteract the accumulation of ROS and are part of the defense reaction. The aim of the present study was to investigate the compartment-specific importance of ascorbate and glutathione during a virulent and avirulent Pseudomonas syringae infection in Arabidopsis thaliana. Peroxisomes were found to be the hotspot for glutathione accumulation reaching 452% and 258% of control levels 24 h postinoculation during the virulent and avirulent infection, respectively. An accumulation of ascorbate could also be observed in vacuoles during Pseudomonas syringae infection, whereas glutathione remained absent in this cell compartment. Neither glutathione nor ascorbate accumulated in the apoplast during pathogen infection demonstrating an only negligible role of these antioxidants in the apoplast during pathogen infection. Compartment-specific changes followed a recently proposed stress model with an increase of ascorbate and glutathione in most cell compartments at the early stages of infection and a strong drop at the later stage of infection when a strong accumulation of ROS and symptoms occurred in the leaves. This study highlights the importance of certain cell compartments and antioxidants in general for the protection of pathogen-induced ROS accumulation.