Introduction: The purpose of this study was to evaluate the effect of final irrigation with a new endodontic irrigant, QMix 2in1 (Dentsply Tulsa Dental, Tulsa, OK), on sealer penetration into dentinal tubules using confocal laser scanning microscopy. Methods: Thirty recently extracted human maxillary incisors were used in the present study. The teeth were instrumented with sodium hypochlorite (NaOCl) irrigation and then divided into 3 groups according to the final irrigation regimen used: (1) control group, 2.5% NaOCl; (2) EDTA + CHX group, 17% (EDTA) followed by 2% chlorhexidine (CHX); and (3) QMix group, QMix 2in1. All teeth were dried and then obturated with gutta-percha and rhodamine B. labeled AH26 sealer (Dentsply DeTrey, Konstanz, Germany). After setting, the roots were sectioned horizontally 3, 5, and 8 mm from the root apex. Sealer penetration into the dentinal tubules was examined by confocal laser scanning microscopy. Results: In the apical sections of the roots, no significant difference was detected in the percentage of sealer penetration between any of the final irrigation groups (P > .05). Both the EDTA + CHX and QMix groups showed significantly more sealer penetration than the control group in the middle and coronal sections. However, no difference was found between the EDTA + CHX and QMix groups in either of these areas (P>.05) Conclusions: Use of EDTA + CHX or QMix during final irrigation significantly improved sealer penetration when compared with the control group in the middle and coronal sections of the roots; however, no effect was observed in the apical sections.