Bacterial infection caused by pathogenic bacteria has long been an intractable issue that threatens human health. Herein, the fact that nanocatalysts with single iron atoms anchored in nitrogen-doped amorphous carbon (SAF NCs) can effectively induce peroxidase-like activities in the presence of H2O2, generating abundant hydroxyl radicals for highly effective bacterial elimination (e.g., Escherichia coli and Staphylococcus aureus), is reported. In combination with the intrinsic photothermal performance of the nanocatalysts, noticeable bacterial-killing effects are extensively investigated. Especially, the antibacterial mechanism of critical cell membrane destruction induced by SAF NCs is unveiled. Based on the bactericidal properties of SAF NCs, in vivo bacterial infections propagated at wounds by E. coli and S. aureus pathogens can be effectively eradicated, resulting in better wound healing. Collectively, the present study highlights the highly efficient in vitro antibacterial and in vivo anti-infection performances by the single-iron-atom-containing nanocatalysts.