<jats:title>Abstract</jats:title><jats:p>Tricalcium silicate (TCS)-based materials produce calcium hydroxide as a byproduct of their hydration reaction. The present study investigated whether calcium ion release (CIR) affects their biological and antimicrobial properties when used as pulp protection materials. The effect of incorporation of micro-silica and calcium phosphate monobasic to radiopacified TCS-based materials was investigated. The commercial TCS-based Biodentine, Bio-C Pulpo, TotalFill Root Repair Material, TheraCal LC and a base/liner- ACTIVA BioACTIVE (Activa) were also evaluated. The hydration and CIR were monitored and correlated with biocompatibility and antimicrobial assessment of eluates. Overall, the additives altered the hydration and leaching profile of the prototype cements. The micro-silica inclusion resulted in a decreased long-term calcium hydroxide formation which was associated with neutralised cytotoxicity and antibacterial activity. Calcium phosphate did not alter the leaching profile, although a stronger antibacterial effect was induced. The commercial materials also had different CIR profiles. The water-based ones had higher CIR, and this was associated with stronger antimicrobial effect but not enhanced biological activity. Both TheraCal LC and Activa exhibited poor degree of conversion, low CIR, acceptable biocompatibility and moderate antibacterial activity. A positive correlation of CIR with antibacterial effectiveness was observed (0.3 < r < 0.49; p = 0.021, p = 0.011 for the two test bacterial cultures). No relation was shown between CIR and cytotoxicity (0.3 < r < 0.49; p = 0.150, p = 0.068 for the two cell cultures studied). The additives modified the CIR. The antimicrobial properties were dependent on the CIR; the cytotoxicity of the materials was unaffected.</jats:p>