The aim of this study was to evaluate the effectiveness of smear layer removal after the use of different irrigation methods (passive ultrasonic irrigation (PUI), continuous ultrasonic irrigation (CUI), apical negative pressure irrigation and conventional irrigation) using scanning electron microscopy (SEM) as an analytical tool. A total of 100 single-canal teeth were decoronated and randomly divided into five groups (n = 20) according to the irrigation method used: conventional irrigation with front outlet syringe, conventional irrigation with lateral outlet syringe, apical negative pressure irrigation (EndoVac), PUI with Irrisafe and CUI with ProUltra PiezoFlow ultrasonic irrigation needle. Root canal preparation was performed with the ProTaper Gold system up to the F4 instrument, and 5.25% NaOCl was used as an irrigant. After chemical-mechanical preparation, the roots were split longitudinally, and the coronal, middle and apical thirds were examined. SEM digital photomicrographs were taken at ×1000 magnification to evaluate the amount of smear layer in each root canal third; CUI significantly removed more smear layer than any other irrigant activation protocol (p < 0.05); CUI was more effective in removing the smear layer than the other irrigation protocols. However, none of the irrigation protocols were able to produce root canals completely free from smear layer.