New bioactive materials have been developed for retrograde root filling. These materials come into contact with vital tissues and facilitate biomineralization and apical repair. The objective of this study was to evaluate the cytocompatibility and bioactivity of two bioactive cements, Bio-C Repair (Angelus, Londrina, Pr, Brazil) and TotalFill BC RRM putty (FGK, Dentaire SA, La-Chaux-de-fonds, Switzerland). The biological properties in human periodontal ligament stem cells (hPDLSCs) that were exposed to Bio-C Repair and TotalFill BC RRM putty were studied. Cell viability, migration, and cell adhesion were analyzed. Moreover, qPCR and mineralization assay were performed to evaluate the bioactivity potential of these cements. The results were statistically analyzed using ANOVA and the Tukey test (p < 0.05). It was observed that cell viability and cell migration in Bio-C Repair and TotalFill BC RRM putty were similar to the control without statistically significant differences, except at 72 h when TotalFill BC RRM putty was slightly lower (p < 0.05). Excellent cell adhesion and morphology were observed with both Bio-C Repair and TotalFill BC RRM putty. Both cements promoted the osteo- and cementogenic differentiation of hPDLSCs. These results suggest that Bio-C Repair and TotalFill BC RRM putty are biologically appropriate materials to be used as retrograde obturation material.