Chitosan hydrogels are biomaterials with excellent potential for biomedical applications. In this study, chitosan hydrogels were prepared at different concentrations and molecular weights by freeze-drying. The chitosan sponges were physically crosslinked using sodium bicarbonate as a crosslinking agent. The X-ray spectroscopy (XPS and XRD diffraction), equilibrium water content, microstructural morphology (confocal microscopy), rheological properties (temperature sweep test), and cytotoxicity of the chitosan hydrogels (MTT assay) were investigated. XPS analysis confirmed that the chitosan hydrogels obtained were physically crosslinked using sodium bicarbonate. The chitosan samples displayed a semi-crystalline nature and a highly porous structure with mean pore size between 115.7 ± 20.5 and 156.3 ± 21.8 µm. In addition, the chitosan hydrogels exhibited high water absorption, showing equilibrium water content values from 23 to 30 times their mass in PBS buffer and high thermal stability from 5 to 60 °C. Also, chitosan hydrogels were non-cytotoxic, obtaining cell viability values ≥ 100% for the HT29 cells. Thus, physically crosslinked chitosan hydrogels can be great candidates as biomaterials for biomedical applications.