Macrophages are essential for the development of innate immune responses against a variety of infectious factors. They detect invading pathogens via their pattern recognition receptors such as toll‐like receptors (TLRs). TLR7/8 recognizes ssRNA from various viruses. In the present study, we have used 2‐DE gel‐based proteomics to find novel TLR7/8 target proteins in human monocyte‐derived macrophages in order to improve our understanding of the virus recognition by this TLR. A total of 27 protein spots were found to be reproducibly differentially expressed between control and TLR7/8 activated 2‐DE gel pairs, 18 spots being more than two‐fold upregulated and nine spots being at least two‐fold downregulated. Several proteins involved in defense against toxic superoxide (O₂⁻) and other reactive oxygen species, such as manganese‐containing superoxide dismutase (SOD2), glutathione peroxidase, and peroxiredoxins 1 and 6 were highly upregulated after TLR7/8 activation. Western blot analysis showed that activation of macrophages with TLR2, TLR3, TLR4, and TLR7/8 ligands also strongly upregulated SOD2 protein expression. In conclusion, our results show that the activation of pattern recognition receptors of the innate immune system results in strong upregulation of SOD2 gene expression suggesting that SOD2 protects macrophages from oxidative stress during microbial infection.