AbstractThe internal topography of the root canal is complex, especially for the mesial root of the permanent first molar. In response to such challenges, enhanced irrigation protocols have been developed, using laser pulses to agitate fluids and enhance the removal of microbial deposits. The aim of this laboratory study was to assess the effectiveness of laser agitation of sodium hypochlorite in removing multispecies biofilms grown in the mesial root of the permanent first molars. The five agitation groups (N = 12 roots for each) were: 940 nm diode laser (superpulsed mode, 50 μs/pulses at 20 Hz using 20 mJ/pulse); 1064 nm Nd: YAG laser (200 μs/pulse at 20 Hz using 20 mJ/pulse); 2940 nm Er: YAG laser (50 μs/pulse at 15 Hz using a 400/14 conical tip in the SWEEPS protocol, with 20 mJ/pulse); passive ultrasonic agitation at 28 kHz (positive control); and irrigation with a 27‐gauge side vented needle for 2 min per canal (negative control). Biofilm removal was assessed by confocal microscopic imaging of root slices at 1, 4 and 7 mm from the root apex. None of the tested methods were effective in completely eradicating biofilm from the most confined regions of the root canal system. The greatest challenge was cleaning the isthmus regions. There was a positive correlation between canal cleaning and isthmus cleaning, suggesting that increased effectiveness in cleaning root canal walls is associated with more effective isthmus cleaning. Wider and narrow isthmuses were cleaned better than long and narrow isthmuses.