A comprehensive research was conducted so as to compare the mechanical, optical, and water absorption properties of transparent, biocompatible, and biodegradable chitosan-chitin nanofiber (ChNF) bio-nanocomposites, prepared by acetic acid, uncross-linked films, and adipic acid, physically cross-linked (uncured) and chemically cross-linked (cured) films, using solution casting. For this purpose, ChNFs were, firstly,first isolated from shrimp shellshells, through various chemo-mechanical treatments. Chemical characterization and crystallization results revealed that the isolated ChNFs contained 88.02 % chitin fibers, with the crystallinity of 84 %. Spectroscopy analysis also confirmed the successful isolation of chitin fibers after chemo-mechanical treatments. In addition, microscopic images shed light on not only the formation of homogenous ChNFs, with the average diameter of about 21 nm, but also depicted the uniform dispersion of ChNFs into the chitosan biofilms containing 5 wt% nanofibers. Moreover, while ultimate tensile strength (UTS) and Young’s modulus (YM) of unplasticized chitosan-acetic acid biofilms were increased with loading ChNF up to 5 wt%, elongations at break (EB) of them were declined, which this. This decrement was then ameliorated using glycerol as a plasticizer. The UTS results of plasticized chitosan-adipic acid biofilms containing physical and chemical crosslinks were respectively increased about 224% and 283%, in comparison with uncross-linked biofilms.