Calcium silicate (CaSi) materials have been used for bone repair and generation due to their osteogenic properties. Tailoring the surface chemistry and structure of CaSi can enhance its clinical performance. There is no direct comparison between microscale and nanoscale CaSi particles. Therefore, this article aimed to compare and evaluate the surface chemistry, structure, and in vitro properties of microscale CaSi (μCaSi) and nanoscale CaSi (nCaSi) particles synthesized by the sol–gel method and precipitation method, respectively. As a result, the semi-crystalline μCaSi powders were assemblies of irregular microparticles containing a major β-dicalcium silicate phase, while the amorphous nCaSi powders consisted of spherical particles with a size of 100 nm. After soaking in a Tris–HCl solution, the amount of Si ions released from nCaSi was higher than that released from μCaSi, but there was no significant difference in Ca ion release between the two CaSi particles. Compared to microscale CaSi (μCaSi), nanoscale CaSi (nCaSi) significantly enhanced the growth and differentiation of human mesenchymal stem cells (hMSC) and inhibited the function of RAW 264.7 macrophages. In the case of antibacterial activity against Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus), nanoscale nCaSi displayed a higher bacteriostatic ratio, a greater growth inhibition zone and more reactive oxygen species (ROS) production than microscale μCaSi. The conclusion is that nanoscale CaSi had greater antibacterial and osteogenic activity compared to microscale CaSi. Next generation CaSi-based materials with unique properties are emerging to meet specific clinical needs.