The purpose of this study was to determine the effectiveness of laser-activated irrigation by photon-induced photoacoustic streaming (PIPS) in the reduction of Enterococcus faecalis in root canal disinfection, varying laser energy output, and sodium hypochlorite (NaOCl) concentration. For effective removal of the smear layer, the sequence and resting time of the final irrigation steps were modified compared with the standard PIPS protocol. Eighty-six extracted single-rooted teeth were mechanically prepared, sterilized, and inoculated with E. faecalis for 4 weeks. Teeth were divided into 9 groups and treated with an Er:YAG laser using a PIPS 600/9 tip at the following parameters: 10 mJ or 20 mJ, 15 Hz, and a 50-microsecond pulse duration at 0.15 W or 0.3 W average power, respectively. Root canals were irrigated with different concentrations of NaOCl (ie, 1%, 3%, and 5% and activated using the adjusted PIPS protocol). The bacterial count was performed immediately after and 48 hours after decontamination and new incubation on an agar plate. A statistically significant difference in bacterial counts (P < .05) was detected in all groups before and directly after the treatment and in groups treated with 5% NaOCl 48 hours after treatment. Scanning electron microscopic imaging showed an absence of bacteria and biofilm in the scanned areas after treatment with 5% NaOCl. Laser-activated irrigation using 5% NaOCl and a modified PIPS protocol resulted in effective eradication of the bacterial biofilm and removal of the smear layer. •The effective reduction of E. in root canal disinfection was performed using a laser.•The adjustment of the final irrigation sequence in the PIPS is proposed.•Treatment safety is increased using lower laser energy without diminished efficacy.•Three percent NaOCl during instrumentation is recommended to avoid the weakening of dentin.