Chitosan (Ch) is a nontoxic and biocompatible polysaccharide extensively used in biomedical applications. Ch, as a polycation, can be combined with anionic polymers by layer-by-layer (LbL) self-assembly, giving rise to multilayered complexed architectures. These structures can be used in tissue engineering strategies, as drug delivery systems, or artificial matrices mimicking the extracellular microenvironment. In this work, Ch was combined with poly(gamma-glutamic acid) (gamma-PGA). gamma-PGA is a polyanion, which was microbially produced, and is known for its low immunogenic reaction and low cytotoxicity. Multilayered ultrathin films were assembled by LbL, with a maximum of six layers. The interaction between both polymers was analyzed by ellipsometry, quartz crystal microbalance with dissipation, Fourier transform infrared spectroscopy, atomic force microscopy, and zeta potential measurements. Ch/gamma-PGA polyelectrolyte multilayers (PEMs) revealed no cytotoxicity according to ISO 10993-5. Overall, this study demonstrates that Ch can interact electrostatically with gamma-PGA forming multilayered films Furthermore, this study provides a comprehensive characterization of Ch/gamma-PGA PEM structures, elucidating the contribution of each layer for the nanostructured films. These model surfaces can be useful substrates to study cell-biomaterial interactions in tissue regeneration.