Due to the presence of hyaluronic acid (HA) in the human body and the ECM (extracellular matrix) mimicking properties of hydrogels, HA based hydrogels are a promising candidate for tissue engineering applications, in particular approaches designed to create scaffolds for repairing or regenerating neurological defects and diseases. Providing the right mechanical and biological properties, such as matrix stiffness, plays a crucial role in mimicking the damaged tissue with the aim of achieving stimulatory effects and promoting proliferation. The tunability of hydrogel properties can be achieved through the modification of crosslinking density, constituent components, or the oxidation of HA. In the present study, we oxidized hyaluronic acid (HA) using sodium metaperiodate (NaIO4) to obtain oxidized hyaluronic acid (OHA) and thus aldehyde groups. Hydrogels are produced by crosslinking OHA with gelatin through Schiff´s base reaction and the use of the enzyme microbial transglutaminase (mTG). These were modified towards different mechanical responses and the effects on long-term stability by varying the OHA, gelatin or mTG amount. 12 different variations in total were investigated. Compression tests as well as swelling/ degradation studies exhibited the crucial influence of gelatin in these hydrogels. Increasing the amount of gelatin and OHA at the same time leads to higher young´s modulus and improved long-term stability and vice versa. Whereby more reinforcing effects could be assessed with higher gelatin amount. Furthermore, the introduction of higher amounts of mTG, in conjunction with lower OHA: Gel ratios, demonstrated the potential for fine-tuning hydrogel properties in this context. Microstructural analyses revealed that the observed mechanical properties were correlated with crosslinking density and mesh size. To assess the suitability of these distinct hydrogel concentrations as substitutes for the ECM, three hydrogel formulations with different young’s moduli were selected and evaluated through experiments with E18 primary neurons by investigating the neuronal branching and outgrowth potential within the hydrogels. These experiments revealed that optimal neuron survival and development occurred at lower component ratios, coupled with higher levels of crosslinking, resulting in an intermediate stiffness of approximately 0.5 kPa.