Active packaging is designed to extend products shelf life by incorporating active components with biological properties in its structure. The main goal of this research was to develop a biodegradable whey protein isolate (WPI)-based film, incorporated with chitosan nanofiber (CSNF) and cinnamon essential oil (CiEO) (both emulsified and Nanostructured lipid carriers (NLC) form). Then, the physicochemical properties of developed bio-nanocomposite were fully characterized. Both water solubility and the water vapor permeability of WPI film decreased significantly (p < 0.05) by incorporating the CSNF into film structure. The good complexation between WPI and CSNF was confirmed by FTIR. Microstructure revealed that the fiber networks were well distributed throughout the films while the morphological heterogeneity and contributed to the reduction of the tensile strength were evident after addition of CiEO. These obtained results from SEM to be quite in accordance with FT-IR findings that confirmed the incorporation of NLCs into bio-nanocomposite structure have been through physical interactions. The film barrier properties to ultraviolet light were increased by adding all of nano-reinforcements. Moreover, the antibacterial activity of resulting films was enhanced by adding CiEO, especially NLC form. This study introduces a novel ecofriendly bio-nano composite in packaging industries for the shelf life extension of different perishable foods.