Substrates that are simultaneously thin, strong, optically transparent, and biocompatible have diverse applications in a range of fundamental and applied fields. While nature-derived materials offer advantages of sustainability and inherent biocompatibility compared to synthetic polymers, their brittleness and swelling, as well as surface charge and chemical functionalization non-conducive to cell growth, can hinder widespread application. In this work, we discuss the fabrication and systematic characterization of polydopamine-coated chitosan thin films. Chitosan is a widely used, partially deacetylated form of chitin, derived from crustaceans and arthropods. Polydopamine (PDA) is derived from chemistries mimicking mussel foot adhesive proteins. A facile dip-coating process of thin and flexible, uncrosslinked chitosan films in aqueous dopamine solutions leads to dramatic changes in physical and chemical properties. We show how the PDA forms time-dependent assemblies on the film surfaces, affecting surface roughness, hydrophilicity, and mechanical strength. Coating the surface for even a few seconds provides functional changes to the films. Our results shows that the optimal coating time is on the order of few hours, whereby the films are optically transparent with excellent extensibility and Young’s modulus, while further coating reduces the benefits of this surface coating. These materials are biocompatible, serving as substrates for cell adhesion and growth while maintaining good viability. Overall, these findings give insight to the effects of PDA assembly on surfaces, and illustrate how a simple, quick, and robust bioinspired coating process can prime substrates for biomedical applications such as tissue engineering, biosensing, and wound healing.