Introduction: Dicalcium silicate is currently used for vital pulp therapy in dentistry. In this research, a novel dicalcium silicate hydraulic cement is expected to be biocompatible and exhibit low cytotoxicity, making it a promising material for vital pulp therapy. The basis of the cytotoxicity test is the ability of cells to survive in the presence of a given toxic compound. Objective: The objective of this study was to determine the cytotoxicity effect of a novel hydraulic cement dicalcium silicate on fibroblast cells. Methods: NIH3T3 fibroblast cells that had undergone serum starvation for 24 h were given hydraulic cement culture media of novel dicalcium silica with concentrations of 1:1, 1:2, 1:4, and Dulbecco’s Modified Eagle Medium as a negative control. The cytotoxicity effect was tested at 24 and 48 h using the MTT assay. The results were analyzed using a statistical nonparametric Kruskal–Wallis test, followed by post hoc using Mann–Whitney for the 24‑h observation group. In contrast, the 48‑h observation was analyzed using a statistical test, the one‑way analysis of variance parametric test, followed by a post hoc test using a t‑test. Results: There was no significant difference in the value of cell viability in the administration of a novel hydraulic cement dicalcium at various concentrations (1:1, 1:2, 1:4) against NIH3T3 fibroblast cells at 24 h of observation, with a mean of the highest viability value at a concentration of 1:4. At 48 h of observation, there was a significant difference in the value of cell viability in the administration of a novel dicalcium silicate hydraulic cement, at various concentrations (1:1, 1:2, 1:4) to NIH3T3 fibroblast cells, with the highest mean viability value treatment group at a concentration of 1:4. Conclusion: A novel dicalcium silicate hydraulic cement in various concentrations (1:1, 1:2, 1:4) did not have a cytotoxic effect on NIH3T3 fibroblast cells at 24 h and 48 h. The highest NIH3T3 fibroblast cell viability value at 24 and 48 h was observed at a concentration of 1:4. © 2025 Journal of Conservative Dentistry and Endodontics.