The aim of this study was to comprehensively evaluate the effects of two main drying techniques – air-drying and freeze-drying – on the physicochemical and biological properties of chitosan-based matrices (chitosan/curdlan and chitosan/agarose) in order to identify the drying method that is more favorable to cell adhesion and proliferation. Freeze-dried matrices exhibited a highly porous, foam-like microstructure with significantly higher surface roughness (Sa: 24.6–50.3 μm) compared to thin, smooth air-dried films (Sa: 2.5–9.9 μm). Water contact angle analysis of rehydrated samples revealed superhydrophilicity in freeze-dried matrices (∼31.4° or undetectable), whereas air-dried matrices remained moderately hydrophilic (54.4–73.6°). Surface chemistry analysis indicated higher amounts of polar species in freeze-dried samples. Both matrices showed gradual biodegradation over time. Protein adsorption analysis revealed that freeze-dried matrices adsorbed significantly more proteins from blood plasma than air-dried matrices. Unexpectedly, freeze-dried matrices hindered cell adhesion and proliferation, in contrast to air-dried matrices. These findings demonstrate that drying conditions can critically determine not only physicochemical properties but also biological performance. This knowledge provides a practical framework for tailoring chitosan-based biomaterials either to support cell growth in regenerative scaffolds or to suppress cell colonization in wound dressings and temporary implants.