The present study aims to explore the cytotoxicity, physicomechanical, thermal, and barrier properties of Juglans regia leaf fiber (J) reinforced PHB-based films, with a focus on evaluating their suitability for biomedical applications. In this work, scaffolds are developed by incorporating varying concentrations (0.5%, 1%, 1.5%, 2% and 2.5%) of J into poly(hydroxybutyrate)/poly(vinylacetate) matrix by solvent casting. These are characterized through Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), thermo-gravimetric analysis (TGA), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA). The results indicated that the sample containing 1.0% (by weight) J (PJ1.0) results in maximum values of the tensile strength (25 MPa) and storage modulus (1.61 GPa) at – 20 °C. Moreover, this sample exhibited favorable thermal, water barrier, and wettability properties. The hydrolytic degradation behavior of the composites is also studied at pH 7.4 and 37 °C for 16 weeks. It is observed that PJ1.0 degrades by 45%, whereas PHB experiences 18% degradation. Furthermore, the cytotoxic nature of the scaffolds is also assessed using C2C12 mouse skeletal muscle cell lines. The results confirmed that PJ1.0 does not show any cytotoxic effects when compared to pure PHB. Thus, findings of this study suggested the potential of Juglans regia fiber for the development of sustainable and mechanically robust materials for biomedical applications.