This study investigated the antibacterial efficacy and cleaning capacity of the Er,Cr:YSGG laser in root canal disinfection.Fifteen teeth were inoculated with an Enterococcus faecalis biofilm and assigned to three groups as follows: Group 1 (control: biofilm only), Group 2 (Ultrasound [US] + 5.25% NaOCl), and Group 3 (Er, Cr at 1.25W, 40 Hz, with a steriline saline water-air spray 30-90%). Samples were taken from the root canals, and two from each group were prepared for a permeability experiment, where 2% methylene blue was placed inside the conduits and then incubated at 37°C for 20 min. The roots were sectioned into cervical, middle, and apical thirds and viewed with an optical microscope to observe dye penetration. The remaining samples (n=3) were prepared for SEM to analyze dentinal tubule permeability. Optical density was analyzed before and after the treatments. Normality was assessed using the Shapiro-Wilk test, and data were analyzed using Student's t-test and ANOVA with a significance level of 5%.Group 2 had a greater colony forming unit reduction with no difference from Group 3. Group 3 had greater methylene blue penetration and dental tubule cleaning. Both methods are effective in bacterial reduction; however, the reduction was greater with 5.25% NaOCl and ultrasound.Er,Cr:YSGG laser significantly removes the smear layer. Key words:Er, Cr: YSGG, Ultrasound, Dentinal tubules, SEM, E. faecalis.