Since it is known that various cell lines may express different behaviours on the scaffolds surface, a comprehensive analysis using various cellular models is needed to evaluate the biomedical potential of developed biomaterials under in vitro conditions. Thus, the aim of this work was to fabricate bone scaffolds composed of a chitosan-agarose matrix reinforced with nanohydroxyapatite and compare the biological response of two cell lines, i.e. mouse calvarial preosteoblasts (MC3T3-E1 Subclone 4) and human foetal osteoblasts (hFOB 1.19). Within this study, the osteoblasts number on the scaffold surface and the osteogenic markers level produced by MC3T3-E1 and hFOB 1.19 cells were determined. Furthermore, changes in calcium and phosphorous ions concentrations in the culture media dedicated for MC3T3-E1 and hFOB 1.19 were estimated after the biomaterial incubation. The obtained results proved that the fabricated biomaterial is characterized by biocompatibility and osteoconductivity since it favours osteoblasts attachment and growth. It also supports the production of osteogenic markers (collagen, bALP, osteocalcin) by MC3T3-E1 and hFOB 1.19 cells. Interestingly, the developed biomaterial exhibits different ion reactivity values in the two culture media dedicated for the mentioned cell lines. It was also revealed that mouse and human osteoblasts differ in the cellular response to the fabricated scaffold. Thus, the use of at least two various cellular models is recommended to carry out a reliable biological characterization of the novel biomaterial. These results demonstrate that the tested bone scaffold is a promising biomaterial for bone regeneration applications, however further biological and physicochemical experiments are essential to fully assess its biomedical potential.