Background: The aim of the study is to determine the cytotoxic, genotoxic and inflammatory effects of indocyanine green (ICG) mediated photodynamic therapy (PDT) in direct contact with L-929 mouse fibroblast cells and over a dentin barrier. Methods: Eight groups were evaluated; control (C), group with a dentin barrier (D), ICG applied directly on the cells (ICG), ICG applied over a dentin barrier (D-ICG), only laser applied (L), laser applied over a dentin barrier (D-L), ICG and laser applied directly on the cells (ICG-L), ICG and laser applied over a dentin barrier (D-ICG-L). Cell viability was evaluated via ATP Assay, DNA damage was evaluated via Comet Assay, and inflammatory markers IL-1β and TNF-α were assessed via ELISA test. Results: Cell viability decreased in group ICG (p<0.001). Cell viability decrease was higher in Group ICG-L (p<0.001). Cell viability decrease was lower in group D-ICG-L (p>0.05). Group L caused an increase in cell number (p<0.001). DNA damage was observed in ICG, D-ICG, and ICG-L groups (p<0.05). None of the groups displayed an increase of inflammatory markers IL-1β and TNF-α (p>0.05). Conclusions: The presence of dentin between ICG and cells acted as a barrier and protected the cells. ICG-mediated PDT did not cause any cytotoxic, genotoxic or inflammatory effect. The use of ICG-mediated PDT for cavity disinfection is acceptable, but at this concentration its use in periodontal pocket disinfection is not recommended due to its cytotoxic and genotoxic properties.