Barrier membranes are used in periodontal applications with the aim of supporting periodontal regeneration by physically blocking migration of epithelial cells. The present work proposes the combination of chitosan (CHT) with bioactive glass nanoparticles (BG-NPs) in order to conceive a novel guided tissue and bone regeneration membrane, fabricated by solvent casting. The CHT/BG-NPs nano composite membranes are characterized in terms of water uptake, mechanical tests, in physiological simulated conditions and in vitro bioactivity tests. The addition of BG-NPs to CHT membranes decreased the mechanical potential of these membranes, but on the other hand its bioactivity is improved. The membranes containing the BG-NPs induced the precipitation of bone-like apatite in simulated body fluids (SBF). Biological tests were carried out using human periodontal ligament cells and human bone marrow stromal cells. CHT/BG-NPs composite membranes showed to promote cell metabolic activity and mineralization. The results indicated that the CHT/BG-NPs composite membranes could potentially be used as temporary guided tissue regeneration membrane in periodontal regeneration, with the possibility to induce bone regeneration.