The current research focuses on the findings of an investigation on optimizing of an electrospun antibacterial gelatin nanocomposite membrane for bone tissue engineering applications. For this reason, soluble starch coated silver nanoparticles (Ag-NPs) and bioactive glass particles (BG) were incorporated in to gelatin (Gt) to fabricate Gt/Ag-NPs/BG nanocomposite membranes. Employing Box-Behnken design, second-order models have been successfully obtained to evaluate the statistical significance of individual and interaction effects of applied voltage, tip-to-collector distance (TCD), and flow rate on fiber diameter. There was a reasonable agreement between the regression R-2 value (0.9542), R-2 predicted value (0.8768), and the R-2 adjusted value (0.9286) across the entire factor space with identical observations for the experimental and model values. Under optimum conditions (applied voltage of 26 kV, TCD of 180 mm, and flow rate of 0.5 mL/h), the nanocomposite membrane with similar fiber size of bone tissue extracellular matrix can be fabricated with the predicted value of 557 nm obtained by the proposed model. The optimized nanofiber membrane was fabricated under these conditions and average fiber diameter of this membrane was found as 472 +/- 94 nm. The characterization studies of this nanofiber suggest that obtained nanocomposite is a potential candidate for bone tissue engineering applications.