The Fabrication of appropriate skin wound dressing is a promising strategy to treat patients with severe skin injuries. In recent years, significant effort is invested in nanocomposite films made from natural polymers that have a lot of potential as artificial scaffolds for wound healing. Thus, the aim of this research was to produce nanocomposite film based on dialdehyde-corn starch/gelatin/bacterial nano-cellulose containing ZnO-loaded halloysite nanotubes (DAS-G/BC/ZHal) for tissue engineering applications. The synthesized films were examined for thermal stability, swelling ratio, degradation, and mechanical properties. The results revealed that by increasing the amount of BC (from 0.03 wt% to 0.12 wt%) in the films; the thermal stability improved. Also, the synthesized ZHal were incorporated into the DAS-G/BC nanocomposite films as a reinforcing filler and a good antibacterial agent with different concentrations of ZHal (3 and 5 wt%). Moreover, the produced DAS-G/BC/ZHal nanocomposite films significantly (P < 0.05) improved cell growth of NIH-3 T3 fibroblast cells and proliferation (around 150%) in the presence of BC and ZHal which revealed that these nanocomposite films are cytocompatible. The antibacterial activity was evaluated against Escherichia coli (E.Coli), and Staphylococcus aureus (S.aureus), which are the pathogens bacteria detected in wound infections. According to the obtained results, the generated films showed a suitable biomedical scaffold with excellent performance in tissue engineering applications.