The ability of wound dressing materials to tackle skin pathogens colonization that is associated with open wound infections is limited. Recently, green-synthesized metal oxide nanoparticles has received a lot of attention to overcome this limitation. However, titanium dioxide nanoparticles ( TiO2-NPs) exhibit exceptional antibacterial properties. In this work, several concentrations (0, 1, 3, and 5 wt.%) of TiO2 NPs prepared using Aloe vera leaf extract were added to a blend of polyvinyl alcohol and sodium alginate (PVA:SA). This nanocomposite was designed to enhance the healing process of wounds. The interaction between the PVA:SA composite and the TiO2 NPs was confirmed by FTIR. The thermal behavior of the nanocomposite films was investigated using DSC and TGA. The experimental results indicate that the glass transition temperatures of the nanocomposites increased by increasing the added amount of TiO2 NPs to be 53.7 °C (1 wt.%), 55.8 °C (3 wt.%), and 60.6 °C (5 wt.%), which were consistently lower than the glass transition temperature of the matrix material (69.6 °C). The Dynamic Mechanical Analysis was examined. The nanocomposite doped with 5 wt.% of TiO2 NPs detected a high storage modulus (21.6 × 108). Based on swelling and degradation studies, the prepared PVA:SA:TiO2 nanocomposite films have an excellent swelling rate, and the inclusion of TiO2 NPs increases the stability of the polymeric matrix. The PVA:SA:TiO2 nanocomposite films exhibited a superior antibacterial efficacy against Gram-positive bacteria such as Bacillus cereus and Staphylococcus aureus, compared to their effectiveness against Gram-negative bacteria like Escherichia coli. Moreover, the nanocomposite films were biocompatible with Human Skin Fibroblast. Therefore, the developed PVA:SA:TiO2 nanocomposite films suit wound dressing applications.