Objective: To evaluate the effectiveness of two irrigation techniques in eliminating, Enterococcus faecalis and Fusobacterium nucleatum, from the root canal system of extracted single-rooted human teeth. Methods: The irrigation techniques employed were, conventional positive pressure and sonic irrigation testing EndoActivator tip sizes. Immediately after irrigation, real time PCR (qPCR) conducted on paper point samples assessed bacterial DNA. Roots were split longitudinally and one half evaluated under scanning electron microscopy (SEM) to enumerate surface contamination; 1 to 2 mm sections cut perpendicular to the canal were examined by confocal laser scanning microscopy (CLSM) to evaluate tubule decontamination. RESULTS: SEM: There was a statistically significant difference between the experimental groups and the control in the apical third of the canal (p<0.0001). However there was no significant difference between the positive pressure and EndoActivator groups. Tip size and activation time had no significant influence on bacterial removal. CLSM: There were significantly fewer live bacteria in the dentinal tubules of the 15.02/60 second and 35.04/30 second EndoActivator groups than the control group (p<0.01). qPCR: There were significantly more bacteria in the control group (p<0.0001). F. nucleatum was detected in significantly higher number than E. faecalis in the control group (p<0.0001). CONCLUSION: There is no difference between the EndoActivator and positive pressure irrigation in the apical third. The EndoActivator did provide deeper penetration of sodium hypochlorite into the dentinal tubules.