The aim of this study was to develop a functional collagen membrane that is treated with poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with dexamethasone (DEX) as a bioactive molecule for guided bone regeneration (GBR). The DEX-loaded PLGA microparticles prepared using water-in-oil standard emulsion method were precoated with positively charged polyethylenimine molecules and later immobilized onto the surface of the collagen membrane; the microparticles were physically immobilized using counter charges of positively charged PLGA microparticles and the negatively charged collagen membrane surface. The release profile of DEX over a 4-week immersion study indicated an initial burst release followed by a sustained release. The performance of this system was investigated using rats with calvarial bone defects. The in vivo evaluation of the defects filled with membrane containing DEX-loaded PLGA microparticles indicated enhanced volume and quality of new bone formation compared with defects that were either unfilled or filled with membrane alone. This innovative platform for bioactive molecule delivery more potently induced osteogenesis, which may be exploited in implantable membranes for stem cell therapy or improved in vivo performance. In conclusion, this newly developed collagen membrane treated with drug-loaded PLGA microparticles might be applicable as a promising bone graft substitute for GBR.