Raman spectroscopic imaging and mapping were applied to characterise three-compound ceramic composite biomaterial consisting of chitosan, β-1,3-d-glucan (curdlan) and hydroxyapatite (HA) developed as a bone tissue engineering product (TEP). In this rapidly advancing domain of medical science, the urge for quick, reliable and specific method for products evaluation and tissue–implant interaction, in this case bone formation process, is constantly present. Two types of stem cells, adipose-derived stem cells (ADSCs) and bone marrow-derived stem cells (BMDSCs), were cultured on composite surface. Raman spectroscopic imaging provided advantageous information on molecular differences and spatial distribution of compounds within and between the cell-seeded and untreated samples at a microscopic level. With the use of this, it was possible to confirm composite biocompatibility and bioactivity in vitro. Deposition of HA and changes in its crystallinity along with protein adsorption proved new bone tissue formation in both mesenchymal stem cell samples, where the cells proliferated, differentiated and produced biomineralised extracellular matrix (ECM). The usefulness of spectroscopic Raman imaging was confirmed in tissue engineering in terms of both the organic and inorganic components considering composite–cells interaction.