Guided bone regeneration (GBR) membranes have been commonly used for bone defect repair in clinical medicine. However, the existing GBR membranes are often limited by weak mechanical strength, poor antibacterial effect, overquick degradation or non-degradation, etc. Here, inspired by natural nacre and the most commonly used GBR membrane (Bio-Gide), we report a new kind of GBR membrane fabricated by combining evaporation-induced self-assembly with a subsequent ice-templating procedure. Similar to the Bio-Gide membrane, it also consists of a bilayer structure including a compact nacre-like layer and a porous layer. Furthermore, it exhibits high mechanical properties and other functions not achieved by the Bio-Gide membrane. The obtained high mechanical properties and multiple functions, including effective bacteriostasis, appropriate degradation rate, and biocompatibility, are superior to those of previously reported GBR membranes. This multifunctional bilayer membrane, fabricated via a simple and straightforward method, may be an appropriate candidate as a bioactive GBR membrane for clinical application. Display omitted •Evaporation-induced self-assembly and ice-templating technique are combined•A unique bilayer structure is designed to fulfill functional requirement•Nacre-inspired structure endows the GBR membrane with high strength and toughness•Most biological functions for an ideal GBR membrane were achieved Treatment of bone defects by applying a barrier membrane, namely a guided bone regeneration (GBR) membrane, is commonly used in clinical medicine. In contrast to commercially available GBR membranes such as collagen membrane or titanium-reinforced PTFE membrane, which exhibit the problems of weak mechanical strength, poor antibacterial effect, overquick degradation or non-degradation, etc., we demonstrated a multifunctional bilayer nanocomposite GBR membrane fabricated by combining nacre-inspired self-assembly with an ice-templating self-assembly technique. The compact nacre-like layer provides a barrier function while ensuring high strength and toughness of the GBR membrane. The porous layer is designed for promoting osteoblast adhesion. In addition, effective antimicrobial activity, appropriate degradation rate, and favorable biocompatibility are fulfilled, rendering this multifunctional GBR membrane as an appropriate candidate for clinical application. The guided bone regeneration (GBR) technique, based on applying a physical barrier membrane, is one of the most effective approaches to achieve osteogenesis. Inspired by biological functions for an ideal GBR membrane, the bilayer multifunctional GBR membrane designed in this work exhibits superior mechanical properties and relevant biological functions, which is distinct from previously reported GBR membranes and may be an appropriate candidate as a bioactive GBR membrane for clinical application. This would boost the substantial progress of biomedical engineering.