Oxidative stress plays an important role in biology and is also an important factor in the pathogen-host relationship. The pathogenic free-living amoeba Acanthamoeba castellanii has several pathways to cope with reactive oxygen species and the damage they cause. In this study, we aimed to provide a comprehensive analysis of the amoeba's response to different sources of oxidative stress. Using whole-cell proteomic analysis, we obtained a complex picture of the changes in the proteome and identified potential key players in the defense against oxidative stress. Importantly, from the differential proteomics analysis we identified a candidate efflux pump that may be involved in Acanthamoeba drug resistance.