Introduction: This study was to compare the effect of combining agitation techniques on bacterial biofilm eradication to their effect separately. Methods and Materials: Cleaning and shaping was performed on human premolars then divided randomly to four groups: passive syringe irrigation, passive ultrasonic irrigation (PUI) , XP- endo Finisher (XPF) (FKG Dentaire, Swiss Endo, Switzerland) and combined (PUI and XPF). Bacterial biofilm was evaluated using Confocal Laser Scanning Microscope (CLSM). Two-way mixed model ANOVA to evaluate different tested variables along with interactions . Pairwise t-tests with Bonferroni correction compared main and simple effects. The statistical significance level was set at P < 0.05. Results: There was no significant difference on combining agitation techniques (PUI and XPF) in comparison to single technique agitation. PUI showed highest significance in comparison to other irrigation techniques. Regardless of agitation technique apical third showed highest percentage of dead bacterial followed by middle and coronal thirds. Conclusion: Complete eradication of bacterial biofilm is impossible. Regardless of the agitation technique used bacterial elimination is better in comparison to passive syringe irrigation.