Guided tissue regeneration (GTR) membranes have great potential to promote periodontal tissue regeneration and reestablishment. However, the regeneration potential and microbial infection resistance of current GTR membranes still need to be improved. Here, a bi-layered nanofibrous membrane on the basis of poly (lactic-co-glycolic acid) (PLGA)/gelatin with osteogenic and antibacterial functions was fabricated for periodontal tissue regeneration. The antimicrobial layer (AL) of the bi-layered nanofibrous membrane was composed of nanofibrous PLGA/gelatin nanofibers loaded with nano-silver (nAg), while the osteoconductive layer (OL) of the nanofibrous membrane consisted of PLGA/gelatin nanofibers loaded with nano-hydroxyapatite (nHA). The bi-layered nanofibrous membrane was examined by scanning electron microscopy (SEM), energy dispersive spectrometer (EDS), transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectrometry (XPS) and X-ray diffractometry (XRD). The results showed that nHA and nAg particles were well evenly loaded or embedded in PLGA/gelatin nanofibers. The cell culture experiments suggested that the bi-layered nanofibrous membrane possessed good cytocompatibility and the OL of the bi-layered nanofibrous membrane possessed an enhanced osteogenic capacity for human osteoblast-like cells (MG63), which was verified by the good cell viability and the increased alkaline phosphatase (ALP) activity, respectively. The results of in vitro antimicrobial study displayed that the AL of the bi-layered nanofibrous membrane possessed an effective antibacterial capability. In conclusion, the prepared bi-layered nanofibrous membrane with osteogenic and antibacterial functions may have great potential for periodontal tissue regeneration and reestablishment. [Formula: see text]