A composite slurry from silver ion-substituted nano-hydroxyapatite, titania nano-particles and polyamide 66 (Ag-nHA/TiO(2)/PA66) was prepared and used to fabricate a novel antimicrobial membrane with a gradient porous structure for guided bone regeneration (GBR). Subsequently, assays were performed to determine the cytocompatibility, as well as the bone biocompatibility of the prepared membranes. To investigate the cytocompatibility of the Ag-nHA/TiO(2)/PA66 membrane, in vitro studies were done with osteoblast-like cells (MG63) and the viability, alkaline phosphatase activity (ALP) and morphology of cells cultured on the membrane were determined. The bone biocompatibility of the membranes was finally assessed in animal experiments, in which nano-hydroxyapatite/polyamide 66 (nHA/PA66) and pure polyamide 66 (PA66) membranes were compared. The in vitro cell-culture experiments showed that Ag-nHA/TiO(2)/PA66 antimicrobial membrane evoked good cell affinity and cytocompatibility. The in vivo study showed that Ag-nHA/TiO(2)/PA66 asymmetric porous barrier membrane resulted in complete closure of 5-mm bone defects as created in the skull of rats after 8 weeks of implantation. In conclusion, the Ag-nHA/TiO(2)/PA66 membrane has the potential to be applied in GBR, especially in infected tissue or areas with high bacteria concentration.