Background: Calcium silicate-based cements are biomaterials with calcium oxide and carbonate filler additives. Their properties are close to those of dentin, making them useful in restorative dentistry and endodontics. The aim of this study was to evaluate the in vitro biological effects of two such calcium silicate cements, Biodentine (BD) and Bioroot (BR), on dental stem cells in both direct and indirect contact models. The two models used aimed to mimic reparative dentin formation (direct contact) and reactionary dentin formation (indirect contact). An original aspect of this study is the use of an interposed thin agarose gel layer to assess the effects of diffusible components from the materials. Results: The two biomaterials were compared and did not modify dental pulp stem cell (DPSC) proliferation. BD and BR showed no significant cytotoxicity, although some cell death occurred in direct contact. No apoptosis or inflammation induction was detected. A striking increase of mineralization induction was observed in the presence of BD and BR, and this effect was greater in direct contact. Surprisingly, biomineralization occurred even in the absence of mineralization medium. This differentiation was accompanied by expression of odontoblast-associated genes. Exposure by indirect contact did not stimulate the induction to such a level. Conclusion: These two biomaterials both seem to be bioactive and biocompatible, preserving DPSC proliferation, migration and adhesion. The observed strong mineralization induction through direct contact highlights the potential of these biomaterials for clinical application in dentin-pulp complex regeneration.