The guided tissue regeneration (GTR) technique with GTR membranes is an efficient method for repairing periodontal defects. Conventional periodontal membranes act as physical barriers that resist the growth of fibroblasts, epithelial cells, and connective tissue. However, they cannot facilitate the regeneration of periodontal tissue. To address this issue, the exploitation of novel GTR membranes with bioactive functions based on therapeutic requirements is critical. Herein, we exploited a biodegradable bilayer polyurethane fibrous membrane by uniaxial electrostatic spinning to construct two sides with Janus properties by integrating the bioactive molecule dopamine (DA) and antimicrobial Gemini quaternary ammonium salt (QAS). The DA-containing side, located inside the injury, can effectively promote cell adhesion and mesenchymal stem cell growth as well as support mineralization and antioxidant properties, which are beneficial for bone regeneration. The QAS-containing side, located on the outer surface of the injury, endows antibacterial properties and limits fibroblast adhesion and growth on its surface owing to its strong hydrophilicity. An in vivo study demonstrates that the Janus polyurethane fibrous membrane can significantly promote the regeneration of periodontal defects in rats. Owing to its superior mechanical properties and biocompatibility, this polyurethane fibrous membrane has potential applications in the field of periodontal regeneration.