AimTo evaluate in a laboratory setting the antibiofilm activity of several irrigating protocols including conventional irrigation, ultrasonic activation and XP‐endo Finisher, with a mixture of sodium hypochlorite and etidronic acid in infected isthmuses and root canals of extracted human mandibular molar teeth.MethodologyFifty‐six mesial roots of mandibular molars, half of them with a continuous isthmus from the cervical to the apical third between the two root canals (type 1), and the other half with a continuous isthmus from the cervical to the middle third and one canal in the apical third (type 2), were included. The root canals were contaminated for 7 days with an Enterococcus faecalis suspension. There were three experimental groups plus a control group (n = 7 per type of root canal anatomy). All the root canals, except for the control group that was not treated, were chemomechanically prepared and then assigned to one of the experimental groups according to the final adjunctive procedure: conventional irrigation, ultrasonic activation or XP‐endo Finisher activation. The irrigating solution used was a combination of 2.5% sodium hypochlorite and 9% etidronic acid, and the final protocols were applied for three cycles of 30 s with a 3 mL volume. The antibiofilm activity was evaluated at each location (root canal and isthmus) and third (cervical, middle and apical) using confocal laser scanning microscopy and the live/dead technique. Statistical analysis was performed using SPSS (descriptive statistics) and SUDAAN (P‐value calculations).ResultsRoot canals had significantly lower biovolume values than the isthmuses (P < 0.05). The biovolume in the root canals was significantly reduced in all the experimental groups in all the thirds except for conventional irrigation in the apical third (P > 0.05). In the cervical and middle thirds, ultrasonic activation was associated with the lowest biovolumes (P < 0.05), followed by XP‐endo Finisher. In the isthmus, disinfection was similar in all the thirds for all the protocols. Conventional irrigation was associated with intermediate values with no significant differences from the control group or from the activated protocols (P > 0.05), although the latter were significantly different from the control group (P < 0.05). No differences were found between ultrasonic activation and XP‐endo Finisher in the middle and apical thirds (P > 0.05) in the isthmuses.ConclusionsIn this laboratory study on extracted teeth, the isthmus was more difficult to disinfect than root canals. In the root canals, ultrasonic activation and XP‐endo Finisher had a greater effectiveness than conventional irrigation. In the isthmuses, no differences were observed between the two activation techniques and conventional irrigation.