Objective: To develop a clinically relevant complex multi-species biofilm model of endodontic infection and evaluate the performance of new laser technologies in vitro and in vivo. Methods: For the in vitro studies, bacterial species were selected based on the literature and the correlation network analysis (CNA) of our collection of 206 root canal samples. Biofilms were developed for 7 and 14 days. Three tooth models (SM, DMK, SMS) were compared using 200 extracted human premolars and four different laser technologies were tested. For the in vivo studies, patients presenting apical periodontitis were selected for treatment using the standard of care (NaOCl, control) or adjunctive laser treatment (test). Outcomes measured included CFU reduction and pain assessment. Results were evaluated using culture, SEM, FISH and CLSM LIVE/DEAD analysis. Data were analyzed using Kruskal-Wallis, PCA and GEE. Results: Tooth models were assessed with E. faecalis biofilms and tested using a multi-species biofilm composed of A. viscosus, F. nucleatum, P. micra, P. nigrescens and S. sanguinis. DMK was the most effective tooth model. EdgePRO™ was the most effective laser in the presenc of NaOCl and LEAP™ in its absence. Fifty-two patients completed the study. CFUs were reduced by both treatments. No differences were observed regarding post-operative pain. Conclusion: The new multi-species biofilm model developed here is a clinically relevant and effective tool to assess new treatments. EdgePRO™ is effective in reducing CFUs of in vitro and in vivo biofilms.