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Guided bone regeneration (GBR) membranes are commonly used for periodontal tissue regeneration. Due to the complications of existing GBR membranes, the design of bioactive membranes is still relevant. GBR membranes with an asymmetric structure can accommodate the functional requirements of different interfacial tissues. Here, poly(lactic acid-glycolic acid) (PLGA) was selected as the matrix for preparing a bi-layered membrane with both dense and porous structure. The dense layer for blocking soft tissues was incorporated with zinc (Zn) particles, while the porous layer for promoting bone regeneration was co-incorporated with magnesium (Mg) and Zn particles. Mg/Zn-embedded PLGA membranes exhibited 166% higher mechanical strength in comparison with pure PLGA membranes and showed suitable degradation properties with a sequential ion release behavior of Mg2+first and continuously Zn2+. More importantly, the release of Zn2+from bi-layered PLGA endowed GBR membranes with excellent antibacterial activity (antibacterial rate > 69.3%) as well as good cytocompatibility with MC3T3-E1 (mouse calvaria pre-osteoblastic cells) and HGF-1 (human gingival fibroblast cells). Thus, the asymmetric bi-layered PLGA membranes embedded with Mg and Zn particles provide a simple and effective strategy to not only reinforce the PLGA membrane but also endow membranes with osteogenic and antibacterial activity due to the continuous ion release profile, which serves as a promising candidate for use in GBR therapy.