The main objective of this work was to prepare biodegradable packaging film based on the organic base polymer for the replacement of petroleum based plastic films. Therefore, the present investigation is conducted to characterize gluten-polyvinyl alcohol (PVA) based bionanocomposite films containing two concentrations of montmorillonite (MMT) as 2% and 4% and chitosan as 1.5% and 2.5% for their functional properties, barrier properties, morphological, biodegradable, and microbial properties. Results indicated that films prepared with 2% MMT and 2.5% chitosan has represented high transparency with low opacity value (2.47 AUmm(-1)). Further, tensile strength (TS) and water vapour transmission rate (WVTR) of the films were found higher in the same film as 58.94 MPa and 0.06 g/h cm(2), respectively. However, uniform and smooth surface morphology was obtained due to the interaction between MMT (2%) and matrix. While, increasing the concentration of MMT cause cavities on the surface of films and reduced TS of the films. The composite films degraded under soil within 55 days as noticeable achievement in research area. Moreover, intercalation between gluten-PVA matrix and MMT resulted in appreciable modifications of their structures, which were supported by X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy. The nanoclay exfoliates for better interaction with other components and large surface area make their functionality more intense in comparison to macromolecules. Therefore, the synergistic effect of MMT and chitosan has enhanced functional, mechanical and barrier characteristics, can be an alternative approach for the replacement of hazardous non-biodegradable plastic packaging material being used for industrial applications.