Nanozymes with oxidase (OXD)-like activity have emerged as promising antibacterial agents due to their capability of catalyzing atmospheric O to generate highly active free radicals. However, the precise engineering of functional nanozyme at the atomic level for antibacterial therapy presents a challenge. Here, atomically dispersed Fe atoms were loaded onto onion-like carbon (OLC) through a ligand-assisted calcination strategy, yielding a single-atom nanozyme (FeSA-OLC) with enhanced oxidase-like activity. The FeSA-OLC could catalyze the decomposition of O to produce active hydroxyl radicals (·OH) owing to the fully exposed Fe atoms and a highly curved carbon shell. Density functional theory calculation revealed that the single-atom Fe sites facilitated the generation of free radical species by promoting the adsorption and cleavage of OO bond. Meanwhile, the FeSA-OLC exhibited a notable photothermal conversion efficiency of 66.48% under near-infrared laser irradiation. Furthermore, in vitro experimental results demonstrated a synergistic antibacterial effect towards Escherichia coli due to the photothermal-enhanced oxidase-like activity. Overall, this work introduced a strategy to develop OLC-based single-atom nanozyme, thereby offering new avenues for photothermal-augmented antibacterial therapy.