Ca- and P-based bioactive glasses are excellent candidates for design and manufacture of biomaterials. Understanding the structure and physico-chemical–thermal behaviour of bioactive glasses is a fundamental step towards the design of a new generation of biocompatible materials. In this study, the structure of SiO₂–CaO–Na₂O glasses and its derivatives, obtained by substituting Na₂O with P₂O₅ and prepared by melt–quench technique, was studied with neutron and electron diffraction techniques combined with thermal analysis, high-resolution electron microscopy and X-ray photoelectron spectroscopy. Neutron and electron diffraction data were analysed with reverse Monte Carlo simulation and pair distribution function analysis, respectively. Bioactivity of P₂O₅ substituted glasses was also investigated and proven in vitro using simulated body fluid. Based on the structural analysis, it was found that Si and P atoms are in well-defined tetrahedral units with a bond distance of 1.60 Å for both Si–O and P–O bonds, although P exhibits a higher average coordination number than Si. With increasing phosphate content, tendentious changes in the glass behaviour were observed. Linear increase in T_g, supported by the changes in the average coordination numbers of Si and P, indicates strengthening of network structure with increasing P content and formation of P–O–Ca atomic linkages, which lead to Ca–P-rich atomic environments in the silicate network. These Ca–P-rich environments trap volatile elements and thus decrease the total weight loss during heating at higher P concentrations. In the case of the highest investigated P₂O₅ content (5 mol%), nanoscale structural inhomogeneity and the formation of Ca–P-rich clusters were also revealed by electron diffraction and atomic resolution imaging. This type of Ca–(Na)–P clustering has a key role in the behaviour of phosphate-substituted silicate glasses under physiological conditions.