© 2022 Elsevier B.V.The aim of the study was to compare the penetration depth and percentage of Irritrol and chlorhexidine (CHX) solutions into dentinal tubules using conventional needle irrigation (CI), EDDY, and photon-induced photoacoustic streaming (PIPS). Sixty oval shaped mandibular premolar teeth were divided into two main groups according to the irrigating solutions as follows; Irritrol and CHX, which were later randomly divided into three subgroups according to the activation technique; CI, EDDY and PIPS (n = 10). Following the irrigation activation procedures, all roots were horizontally sectioned at 3 (apical), 8 (middle), and 13 (coronal) mm from the root apex and then examined with confocal laser scanning microscope (CLSM). Penetration depth (µm) and penetration percentage (%) as determined from the images were analyzed using LSM Image Browser software (Zeiss) and Image J analysis software (V. 1.44p; National Institutes of Health, Bethesda, MD), respectively. Data were statistically analyzed using Kruskal–Wallis and Mann–Whitney U tests were performed for multiple comparisons. In both Irritrol and CHX groups, the PIPS activation system demonstrated the highest values for irrigant penetration depth and penetration percentage compared with the other activation methods. Activation of the CHX and Irritrol the final irrigating solutions with PIPS activation technique improved the penetration depth and percentage solutions into the dentinal tubules.