Barrier membranes are used in periodontal tissue engineering for successful neo-bone tissue formation and prevention of bacterial colonization. We aimed to prepare and characterize novel 7% boron-modified bioactive glass (7B-BG) containing bilayered membrane for this end. We hypothesized that presence of 7B-BG could promote structural and biological properties of guided bone regeneration (GBR) membrane. Cellulose acetate (CA) layer was prepared by solvent casting, and functionally graded layer of CA/gelatin/BG nanoparticles was prepared by electrospinning. 0B-BG, and 7B-BG were synthesized by quick alkali-mediated sol-gel method and were characterized by scanning electron microscopy (SEM) and Fourier-transform Raman spectroscopy. Membranes were cross-linked with glutaraldehyde to preserve their stability. SEM analysis showed the asymmetric nature of membranes consisting of a smooth membrane layer and a rough surface composed of 0B-BG and 7B-BG containing nanofibres. 7B-BG addition increased surface wettability (from 110.5° ± 0.8 to 73.46° ± 7.6) and biodegradability of the membranes. Additionally, a significant increase in Ca-P layer formation was observed in 7B-BG containing group after 1-week incubation in stimulated body fluid. 7B-BG incorporation resulted in a decrease in tensile strength and Young's modulus values. Human dental pulp stem cells showed better attachment, spreading, and proliferation on 7B-BG containing bilayered membranes. Osteogenic differentiation analysis revealed higher alkaline phosphatase (ALP) enzyme activity of cells (~1.5-fold), higher intracellular calcium deposition (approximately twofold), and higher calcium deposition revealed by Alizarin red staining on 7B-BG containing bilayered membranes. Overall, results suggested that functionally graded bilayered membranes hold potential for GBR applications in regenerative dentistry.