Three‐dimensional (3D) biomimetic cell culture platforms offer more realistic microenvironments that cells naturally experience in vivo. We developed a tunable hyaluronan‐based hydrogels that could easily be modified to mimic healthy or malignant extracellular matrices (ECMs). For that, we pre‐functionalized our hydrogels with an adhesive polypeptide (poly‐l‐lysine, PLL) or ECM proteins (type III and type IV collagens), naturally present in tumorous tissues, and next, we tuned their stiffness by crosslinking with gradual concentrations of genipin (GnP). Then, we thoroughly characterized our substrates before testing them with glioblastoma and breast cancer cells, and thereafter with endothelial cells. Overall, our hydrogels exhibited (a) increasing stiffness with GnP concentration for every pre‐functionalization and (b) efficient enzyme resistance with PLL treatment, and also with type IV collagen but to a lesser extent. While PLL‐treated hydrogels were not favorable to the culture of any glioblastoma cell lines, they enhanced the proliferation of breast cancer cells in a stiffness‐dependent manner. Contrary to type III collagen, type IV collagen pre‐treated hydrogels supported the proliferation of glioblastoma cells. The as‐desired HA‐based 3D tumor‐like models we developed may provide a useful platform for the study of various cancer cells by simply tuning their biochemical composition and their mechanical properties.