Macedo, MargaridaNeves, João AlbernazPérez, Alejandro R.Proença, LuísBessa, Lucinda J.2026-06-082026-06-082026-04Macedo, M., J. Albernaz Neves, A. R. Pérez, L. Proença, and L. J. Bessa. 2026. “ Enhanced Efficacy of Laser-Activated Irrigation (Er,Cr:YSGG) in Eradicating Enterococcus faecalis Biofilm in 3D-Printed Molar Replicas: A Pilot Study.” Clinical and Experimental Dental Research 12: 1–10. https://doi.org/10.1002/cre2.702792057-4347http://hdl.handle.net/10400.26/63545Objectives: This study compared the efficacy of Sonic (EDDY) and Er,Cr:YSGG (2780 nm) laser activation in eradicating Enterococcus faecalis biofilm formed in 3D-printed molar replicas with two mesial canals and one distal canal. Materials and Methods: An in vitro design was implemented using 20 3D-printed mandibular molar replicas mimicking the natural canal morphology. Root canals were inoculated with E. faecalis and incubated for 21 days to allow the development of a mature biofilm. Three irrigation protocols were tested: Conventional needle irrigation (CNI), EDDY sonic activation (SA), and Er,Cr:YSGG (2780 nm) laser activation (LA). The control group was irrigated with phosphate-buffered saline (PBS) without activation. Residual bacterial load was quantified through colony-forming unit (CFU) counts and quantitative PCR (qPCR). Bacterial viability in the apical isthmus was assessed using fluorescence microscopy. A Student's t-test was performed to identify significant differences between CFU/mL values in groups, with significance set at 5% (p < 0.05). Results: CFU counts of E. faecalis were significantly lower in the CNI, SA, and LA groups compared to the control (p < 0.05). In the SA and LA groups, bacterial counts were reduced to the lower detection limit (< log10 CFU/mL of 1.00), suggesting near-total bacterial elimination. qPCR and fluorescence microscopy corroborated these results, providing greater differentiation between the outcomes of sonic and laser activations. Conclusions: Er,Cr:YSGG (2780 nm) laser activation showed superior efficacy in endodontic disinfection by effectively eradicating E. faecalis biofilm, including in the challenging isthmus region, representing a promising method for complex root canal anatomies.eng3D‐printed molar replicasendodontic irrigationEnterococcus faecalis biofilmErCr:YSGG lasersonic activationcontribution to journal10.1002/cre2.70279