Guided tissue regeneration (GTR) is a surgical procedure applied in the reconstruction of periodontal defects, where an occlusive membrane is used to prevent the fast-growing connective tissue from migrating into the defect.In this work, silk fibroin (SF) membranes were developed for periodontal guided tissue regeneration. Solutions of SF with glycerol (GLY) or polyvinyl alcohol (PVA) where prepared at several weight ratios up to 30%, followed by solvent casting and thermal annealing at 85 ºC for periods of 6 and 12 hours to produce high flexible and stable membranes. These were characterized in terms of their morphology, physical integrity, chemical structure, mechanical and thermal properties, swelling capability and in vitrodegradation behavior. The developed blended membranes exhibited high ductility, which is particular relevant considering the need for physical handling and adaptability to the defect. Moreover, the membranes were cultured with human periodontal ligament fibroblast cells (hPDLs) up to 7 days. Also, the higher hydrophilicity and consequent in vitroproteolytic degradability of these blends was superior to pure silk fibroin membranes. In particular SF/GLY blends demonstrated to support high cell adhesion and viability with an adequate hPDLs’ morphology, make them excellent candidates for applications in periodontal regeneration.