Objectives To evaluate the efficacy of a stepwise intraoperative activation (SIA) of irrigants during and after the instrumentation compared with that of a conventional activation (CA) performed only after the instrumentation to remove smear layer and debris using different activation devices. Materials and methods A total of 70 single-rooted teeth were divided into a control group (no activation, n = 10) and two different experimental groups according to the irrigant activation protocol used: group 1 (CA), in which sodium hypochlorite was activated only after the use of the last mechanical file, and group 2 (SIA), in which activation was performed during and after the instrumentation. The two groups were divided into 3 subgroups according to the activation device used (n = 10): passive ultrasonic irrigation (PUI, subgroup a), EndoActivator (EA, subgroup b), and EDDY (subgroup c). The roots were split longitudinally and observed using scanning electron microscopy (SEM) to evaluate the presence of debris and smear layer, and the results were statistically analyzed. Results All activation protocols and devices were more effective than control group in removing smear layer and debris from all root canal thirds (P < 0.05), except for CA-EA (group 1b) in the apical third. In the apical third, SIA was found to be more effective than CA (P < 0.05) to remove smear layer and residual debris when PUI was used, to remove the smear layer when EA was used (P < 0.05), and to remove residual debris when EDDY was used (P < 0.05). PUI and EDDY removed statistically more smear layer and debris than EA in the apical third (P < 0.05). Conclusions The SIA technique improved the smear layer and debris removal from the apical third and debris removal from the coronal third, and PUI and EDDY were more effective than EA in the apical third.