In this work, the potential of titanium dioxide nanoparticles incorporated gellan gum (GG + TiO2-NPs) biofilm as wound dressing material was investigated. The GG + TiO2-NPs biofilm was prepared via evaporative casting technique and was characterized using FTIR, XRD, and SEM to study their physiochemical properties. The mechanical properties, swelling and water vapor transmission rate (WVTR) of biofilm was determined to comply with an ideal wound dressing material. In vitro and in vivo wound healing studies was carried out to evaluate the performance of GG + TiO2-NPs biofilm. In vitro wound healing was studied on 3 T3 mouse fibroblast cells for cell viability, cell proliferation, and scratch assay. The acridine orange/propidium iodide (AO/PI) staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were used to evaluate the viability of cell and cell proliferation. Cell migration assay was analyzed using Essen BioScience IncuCyteTM Zoom system. In vivo wound healing via open excision wounds model on Sprague Dawley rat was studied within 14 days. The FT-IR spectra of GG + TiO2-NPs biofilm show main bands assigned to OH stretching, OH deformation, and TiO stretching modes. XRD pattern of GG + TiO2-NPs biofilm suggesting that TiO2-NPs was successfully incorporated in biofilm and well distributed on the surface as proved by SEM analysis. The GG + TiO2-NPs biofilm shows higher mechanical strength and swelling (3.76 ± 0.11 MPa and 1061 ± 6%) as compared to pure GG film (3.32 ± 0.08 Mpa and 902 ± 6%), respectively. GG + TiO2-NPs biofilm shows good antibacterial properties as 9 ± 0.25 mm and 11 ± 0.06 mm exhibition zone was observed against Staphylococcus aureus and Escherichia coli bacteria, respectively. While no exhibition zone was obtained for pure GG biofilm. GG + TiO2-NPs biofilm also demonstrated better cell-to-cell interaction properties, as it's promoted cell proliferation and cell migration to accelerate open excision wound healing on Sprague Dawley rat. The wound treated with GG + TiO2-NPs biofilm was healed within 14 days, on the other hand, the wound is still can be seen when it was treated with GG. However, GG and GG + TiO2-NPs biofilm show no cytotoxicity effects on mouse fibroblast cells.