Background <p>Dual-functional antibacterial and osteogenic substitutes are advanced tools for treating bone infections while facilitating bone regeneration. These systems incorporate drug delivery mechanisms that allow for the local release of antibiotics and increased treatment efficacy. Decellularized bone extracellular matrix (dECM) offers key advantages for bone xenografts and local drug delivery due to its biocompatibility and low immunogenicity. This study developed a novel scaffold by loading linezolid-poly (lactic-co-glycolic acid) (PLGA-Lin) microparticles onto dECM derived from bovine tibia. The antibacterial activity, cytocompatibility, and osteoconductive properties of the resulting Lin-PLGA/dECM scaffold were assessed in vitro<i>.</i></p> Results <p>The results revealed that the dECM had a porous structure and the Lin-PLGA microparticles infiltrated the scaffold. None of the dECM, PLGA/dECM, or Lin-PLGA/dECM scaffolds had toxic effects on adipose tissue-derived mesenchymal stem cells (ADSCs), while the inhibitory effect of Lin-PLGA/dECM on the <i>Staphylococcus aureus</i> strain was significant. All scaffolds—dECM, PLGA/dECM, and Lin-PLGA/dECM—promoted the bone differentiation of ADSCs, as confirmed by alkaline phosphatase (ALP) and calcium content assays, as well as by the expression of the Runt-related transcription factor 2&#xa0;(Runx2), osteopontin, osteocalcin, and collagen Iα genes.</p> Conclusions <p>The Lin-PLGA/dECM scaffold developed in this study can be considered a biocompatible bone xenograft substitute, as it possesses excellent antibacterial properties and substantial osteogenic potential.</p> Graphical Abstract <p></p>