•3D printing resins have shown similar biological effects to conventional ones.•Freeprint Splint was the most cytotoxic on human gingival fibroblasts of the four dental resin studied.•The results obtained in this study suggest that hGFs showed better in vitro biocompatibility after exposition to Keysplint Soft, being the most eligible dental resin for occlusal devices. : To assess and compare the in vitro biocompatibility of new resins (Keysplint Soft (Keystone Industries), NextDent Ortho Rigid (3D System), and Freeprint Splint (Detax)) and traditional resins (Orthocryl (Dentaurum)) for dental splint. : Standardized discs (n=40) and 1:1, 1:2, and 1:4 extracts of the tested materials were prepared. Human gingival fibroblasts (hGFs) were isolated from gingival tissues. Different biological tests were carried out, including MTT assays to assess cell metabolic activity, cell migration assays, cell cytoskeleton staining, cell apoptosis, generation of intracellular reactive oxygen species (ROS), and scanning electron microscopy (SEM). Statistical analyses were performed using one-way ANOVA and Tukey's post hoc test (p<0.05). : MTT experiments showed that Freeprint Splint significantly reduces the hGF metabolic activity (***p<0.001), whereas SEM analysis showed almost no cells adhered on its surface. Cell migration was significantly lower after exposure to undiluted extracts of Freeprint Splint at 48 and 72h (***p<0.001). Cell cytoskeleton staining assays showed fewer attached cells in 1:1 and 1:2 dilutions of Freeprint Splint. Annexin-V and 7-AAD staining assays showed that only cells exposed to Keysplint Soft extracts displayed similar cell viability to the control group. Finally, ROS levels detected in undiluted extracts of all resins were significantly enhanced compared to the control group (***p<0.001). : The 3D-printed resins and the conventional dental resin showed a similar biocompatibility, except for Freeprint Splint, which was the most cytotoxic on hGFs. : 3D printing has been on the rise in recent years and its use in daily clinical practice is expanding over time. Two of the three 3D-printed resins tested in this study performed as well in the cytotoxicity tests as the conventional one, supporting their use, but caution and further testing are required