Abstract The aims of this study were to evaluate the physical and chemical properties, cytotoxicity and dentinal tubule penetration of a new calcium silicate-based root canal dressing. For pH and calcium ion release evaluation (1, 24, 72 and 168 h) were used a pH meter and colorimetric spectrophotometer, respectively. Radiopacity evaluation followed the ISO 6876:2012. Cytotoxicity was evaluated by the percentage of cell viability using MTT assay. Illustrative images of dentinal tubule penetration were obtained using confocal laser scanning microscopy (CLSM). Data from pH and calcium ion release were statistically analyzed by two-way analysis of variance and Tukey test. Radiopacity was analyzed using the Student t-test. The statistical tests for cytotoxicity results were the one-way analysis of variance and Tukey test. Both materials showed alkaline pH in all experimental times. The pH values for calcium hydroxide paste were higher than bioceramic paste at 1, 24, and 72 h (p<0.05). The calcium ion release of bioceramic was lower than the calcium hydroxide paste only at 24 h (p<0.05). The bioceramic was more radiopaque than the calcium hydroxide paste (p<0.05). Bioceramic paste presented a dose and time-dependent cytotoxic effect after MTT assay. CLSM images showed absence of tubule penetration for both pastes. The new calcium silicate-based canal dressing presented alkaline pH, high calcium release, and acceptable radiopacity. Bio C Temp showed a dose and time-dependent cytotoxic and absence of dentinal tubule penetration.