Abstract Infected bone defects are a common clinical condition, but conventional treatments often fail to achieve the desired outcomes, including addressing antibiotic resistance and preventing nonunion complications. In the presented study, a functionalized decellularized mushroom stem scaffold is developed composed of its naturally aligned channels, Zn 2+ /curcumin MOFs, hydroxyapatite minerals, and icariin. In vitro, It is found that functionalized acellular mushroom stem scaffold can control bacterial infections through Zn 2+ /curcumin MOFs. The naturally aligned channels guide bone mesenchymal stem cells (BMSCs) migration, and the components adsorbed on the acellular substrate further promote the migration of BMSCs. Moreover, these functional components further accelerated the polarization of M2 macrophage and osteogenic differentiation of BMSCs. In vivo, the functionalized decellularized mushroom stem scaffold cleared infected bacteria within 3 days, induced extracellular matrix secretion and alignment, and promoted new bone formation to cover defects within 8 weeks. The functionalized decellularized mushroom stem scaffold provides a promising strategy for treating infectious bone defects.