The goal of this research was the fabrication of chitosan (CT)-tragacanth gum (TG)/SiO2 nanocomposite as a potential use in tissue engineering. For the antibacterial activity induction and stabilization of SiO2 nanoparticles (NPs) in the polymer matrix, SiO2 and green synthesized Ag NPs were hybrid with SiO2/Ag. Then, the obtained hybrids were inserted into the CT-TG blend, separately. The ultrasonic waves were applied, and the films were subsequently constructed by the solution casting technique. Finally, the CT-TG/SiO2 nanocomposites and CT-TG/SiO2@Ag nanocomposite films were studied by the variant techniques. The in-vitro bioactivity examination all samples was performed in a simulated body fluid. It was observed that hydroxyapatite was grown more on the surface of the CT-TG/SiO2@Ag (1/1) nanocomposite film in comparison to the other specimens, due to its high surface area and the presence of many specific functional groups. Furthermore, the antibacterial activity assessment of the all samples were performed against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria via well diffusion method. The higher inhibition zone diameters for the CT-TG/SiO2@Ag (1/1) nanocomposite film against both bacteria were observed in the presence of aqueous solution of acetic acid 1% v/v (11 and 13 mm) and deionized water (18 and 20 mm), respectively.