The current single-atom catalysts (SACs) for medicine still suffer from the limited active site density. Here, we develop a synthetic method capable of increasing both the metal loading and mass-specific activity of SACs by exchanging zinc with iron. The constructed iron SACs (h3-FNC) with a high metal loading of 6.27 wt% and an optimized adjacent Fe distance of ~ 4 Å exhibit excellent oxidase-like catalytic performance without significant activity decay after being stored for six months and promising antibacterial effects. Attractively, a “density effect” has been found at a high-enough metal doping amount, at which individual active sites become close enough to interact with each other and alter the electronic structure, resulting in significantly boosted intrinsic activity of single-atomic iron sites in h3-FNCs by 2.3 times compared to low- and medium-loading SACs. Consequently, the overall catalytic activity of h3-FNC is highly improved, with mass activity and metal mass-specific activity that are, respectively, 66 and 315 times higher than those of commercial Pt/C. In addition, h3-FNCs demonstrate efficiently enhanced capability in catalyzing oxygen reduction into superoxide anion (O2·-) and glutathione (GSH) depletion. Both in vitro and in vivo assays demonstrate the superior antibacterial efficacy of h3-FNCs in promoting wound healing. This work presents an intriguing activity-enhancement effect in catalysts and exhibits impressive therapeutic efficacy in combating bacterial infections.