Members of the evolutionary conserved Sco protein family have been intensively studied regarding their role in the assembly of the mitochondrial cytochrome c oxidase. However, experimental and structural data, specifically the presence of a thioredoxin-like fold, suggest that Sco proteins may also play a role in redox homeostasis. In our study, we addressed this putative function of Sco proteins using Saccharomyces cerevisiae as a model system. Like many eukaryotes, this yeast possesses two SCO homologs (SCO1 and SCO2). Mutants bearing a deletion of either of the two genes are not affected in their growth under oxidative stress. However, the concomitant deletion of the SOD1 gene encoding the superoxide dismutase 1 resulted in a distinct phenotype: double deletion strains lacking SCO1 or SCO2 and SOD1 are highly sensitive to oxidative stress and show dramatically increased ROS levels. The respiratory competent double deletion strain Δsco2Δsod1 paved the way to investigate the putative antioxidant function of SCO homologs apart from their role in respiration by complementation analysis. Sco homologs from Drosophila, Arabidopsis, human and two other yeast species were integrated into the genome of the double deletion mutant and the transformants were analyzed for their growth under oxidative stress. Interestingly, all homologs except for Kluyveromyces lactis K07152 and Arabidopsis thaliana HCC1 were able to complement the phenotype, indicating their role in oxidative stress defense. We further applied this complementation-based system to investigate whether pathogenic point mutations affect the putative antioxidant role of hSco2. Surprisingly, all of the mutant alleles failed to restore the ROS-sensitivity of the Δsco2Δsod1 strain. In conclusion, our data not only provide clear evidence for the function of Sco proteins in oxidative stress defense but also offer a valuable tool to investigate this role for other homologous proteins.