AbstractAimThis study evaluated the transdentinal cytotoxic effects of enzymatic agents (EA) for chemomechanical carious tissue removal on human dental pulp cells.MethodologyThe groups were based on the performed dentine treatments (n = 8): G1: Positive Control (PC ‐ no treatment); G2: Negative Control (NC ‐ 35% H₂O₂ for 2 min); G3: Brix 3000™ (BX) for 30 s; G4: BX for 2 min; G5: Papacarie Duo™ (PD) for 30 s; G6: PD for 2 min. The cells were evaluated for viability (VB; MTT assay) and production of reactive oxygen species (ROS; DCFH‐DA assay) and nitric oxide (NO; Griess reagent). A scanning electron microscope provided morphological chemical analyses and energy‐dispersive X‐ray spectroscopy. The data were submitted to the one‐way anova statistical test complemented by Tukey (p < .05).ResultsCell viability decreased by 21.1% and 58.4% in G5 and G6, respectively. ROS production in G3 and G4 maintained basal levels but increased by 171.2% and 75.1% in G5 and G6, respectively.ConclusionsThe Brix3000™ enzymatic agent did not cause indirect cytotoxic effects on pulp cells, regardless of the application time. Conversely, Papacarie Duo™ reduced viability and increased ROS production by pulp cells.