In this study, a novel antibacterial agent was developed based on chitin nanofibrous microspheres loaded with AgNPs and Fe3O4 nanoparticles (Ag-Fe3O4-NMs) for synergistic antibacterial activity and wound healing. Ag-Fe3O4-NMs was prepared via an in situ synthetic method which showed an excellent porosity and wettability. Moreover, Ag-Fe3O4-NMs were capable of sustained release of Ag+ and catalysed the decomposition of low H2O2 concentrations to generate hydroxyl radical (•OH). The •OH and Ag+ showed higher antibacterial activity and inhibited the toxicity with high dose of AgNPs and H2O2. In vitro biocompatibility results suggested that Ag-Fe3O4-NMs have low toxicity and low hemolysis. Thus, a novel antibacterial agent with enhanced synergistic antibacterial activity was obtained by combination of Ag-Fe3O4-NMs and H2O2 at a low and biologlically safe dosage, which could facilitate fibroblast growth, accelerate epithelialization, and promote the healing rate of infected wounds.