The aim of this study was to analyse the cytocompatibility of Surefil One (SuO) with respect to the release of monomers from the material. The following reference materials were chosen: SDR Flow Plus (SDR, Dentsply Sirona, Konstanz, Germany), One Q Bond (Q, Dentalica, Milan, Italy), and Ketac (K, 3M-ESPE, USA). Fifteen dentin discs (2 mm thickness and diameter) were obtained from 15 third molars and were used in this study. After dentin disc permeability measurement, murine fibroblasts were grown, and the pulp surface of the dentinal disc was placed in direct contact with the cells immersed in DMEM. The experimental materials were positioned on the occlusal side of each dentinal disc until a uniform thickness of 2 mm was obtained. Then, the discs were inserted into an artificial pulp chamber for 24 hours to assess the cytocompatibility. Afterwards, the moles of monomers leached from the specimens in DMEM were determined using HPLC. Statistical analysis was performed using ANOVA ( p < 0.05 ). Under the experimental conditions, the toxic effect induced by all tested materials was slight or absent. Diurethane dimethacrylate and acrylic acid were not found in the culture media. It is concluded that all materials have good cytocompatibility consistent with the nondeterminability of the monomers released after polymerization.