This work aimed to compare the biomedical potential of thermally obtained hydrogels consisting of curdlan and a curdlan–whey protein isolate mixture. The purpose of this paper was also to investigate and explain the mechanisms responsible for their different cyto- and biocompatibility. To do this, mechanical and structural properties, wettability, and protein adsorption of biomaterials were analysed and compared. Biological experiments in vitro were carried out on normal human chondrocytes, osteoblasts, and macrophages. Moreover, in vivo studies were performed using the Danio rerio (zebrafish). Our studies showed that in comparison to the curdlan/whey protein isolate hydrogel, the curdlan hydrogel had lower mechanical properties. Its surface was less rough and more hydrophobic and adsorbed a greater amount of blood proteins, but most probably induced their conformational changes. In addition, the curdlan hydrogel exhibited lower cytocompatibility towards normal human osteoblasts and chondrocytes. It also induced greater inflammation in human macrophages in vitro and did not enhance the re-growth of the amputated caudal fin in the zebrafish larvae model when compared to the curdlan/whey protein isolate hydrogel. Hence, our studies demonstrated that a commonly used curdlan hydrogel has a significantly lower biomedical potential compared to the curdlan/whey protein isolate hydrogel proposed by us.