OBJECTIVE: To investigate the influence of sonic agitation or laser-activated irrigation techniques on the removal chlorhexidine (CHX) and modified triple antibiotic paste (mTAP) on the sealer penetration depth and dislocation resistance of Guttaflow Bioseal. METHODS: Single-rooted mandibular premolars (n=96) were prepared with rotary nickel titanium instruments and randomly divided into two groups (n=48) based on the intracanal medicaments used: Group 1, mTAP; Group 2, CHX gel. After 7 days, the specimens in each group were divided into three subgroups (n=16) based on the supplementary irrigation technique used to remove the medicaments: laser activated irrigation (Er, Cr: YSGG laser, Waterlase MD, Biolase Technology Inc., San Clemente, CA, USA), sonic agitation (EndoActivator, Dentslpy Sirona Endodontics, PA, USA) and syringe-and-needle irrigation (control) techniques. Canals were filled with single matched-taper gutta-percha cone and a calcium silicate-based sealer (GuttaFlow® Bioseal, Coltène/Whaledent, Langenau, Germany). At the end of three weeks, sealer penetration was investigated using confocal microscopy (n=6), and dislocation resistance was calculated by measuring the push-out bond strength (n=10). Statistical analysis was performed using three-way analysis of variance (ANOVA) and Tukey post-hoc test (P=0.05). RESULTS: Laser activated irrigation resulted in significantly higher depth of sealer penetration compared to sonic agitation and syringe irrigation (P<0.01). The average sealer penetration depths were recorded as 846.6 µm, 786.5 µm and 505 µm in the Er,Cr: YSGG laser, EndoActivator and control groups, respectively. The mean bond strength obtained in group 3 (syringe-and-needle irrigation) was significantly less than the other groups (P<0.05). The mean values were 9.08 in the Er,Cr: YSGG laser group, 8.44 in the EndoActivator group and 5.08 in the needle group. CONCLUSION: Er,Cr;YSGG laser irrigation to remove the medicaments was advantageous to other irrigation techniques in sealer penetration and dislocation resistance of the sealer.