Aim: To determine the efficacy of sonic or Er,Cr:YSGG laser activation of irrigation protocols on Enterococcus faecalis biofilms in vitro. Materials and methods: E. faecalis biofilms were generated in instrumented root canals (n = 120), and randomly divided into six groups (n = 20) based on the irrigation/activation protocol. Three percent sodium hypochlorite (NaOCl) was used during instrumentation. The six groups included G1: 3% NaOCl /sonic activation; G2: 3% NaOCl/Er,Cr:YSGG laser activation; G3: 17% EDTA- 3% NaOCl/ sonic activation; G4: 17% EDTA- 3% NaOCl/Er,Cr:YSGG laser activation; G5: 3% NaOCl; and G6: saline. Bacterial viability was assessed by confocal microscopy. Dentin powder was obtained for analysing the colony forming units (CFU/ml). Data were analysed by appropriate statistical analyses with P = 0.05. Results: The biofilm along the root canal wall was completely destroyed by all groups except saline. G4 (EDTA-NaOCl / Er,Cr:YSGG activation) showed maximum bacterial destruction within the dentinal tubules of 200 μm depth (P 0.05). At 400 μm within the dentinal tubules, there was no significant difference between the groups (P > 0.05). Culture analysis showed no growth in any of the groups at 200 μm except the saline control. Conclusions: Activation of irrigants using Er,Cr:YSGG laser brought about a significant reduction of bacteria within the dentinal tubules at 200 μm depth. Keywords: biofilm, confocal microscopy, Enterococcus faecalis, laser, sodium hypochlorite, sonic