Objective: The aim of the present research was to evaluate in vitro the antimicrobial efficacy of a new sonic system (EndoActivator) and its capability to promote cleaning, disinfection and removal of an induced biofilm of a virulent strain of E. faecalis. Materials& Methods: The E.faecalis strain was isolated from necrotic pulps from twenty five patients diagnosed with chronic periodontitis with periapical radiolucency. Seventy single rooted teeth were selected for the study. After decoronation, the working length was standardized and the root canals were prepared up to size F4 ProTaper rotary system. .Each root canal was filled with the E. faecalis suspension that was adjusted for 1 McFarland turbidity standard, and then submersed in the prepared bacterial suspension in sterile coded sealed flasks incubated for one week and the presence of biofilm was confirmed using scanning electron microscope (SEM).The bacterial samples were collected before and after treatment, and the colony forming units (CFU) that were grown onto (BHI) plates were counted and the results were evaluated. Results: The present study showed that the difference between standard syringe irrigation and EndoActivator was statistically non-significant (p>0.05), although the absolute mean bacterial count resulting from using EndoActivator group was slightly higher than standard syringe irrigation. There was a significance increase of bacterial reduction achieved by irrigation with NaOCl and CHX more than saline (P<0.05).There was no significant difference between NaOCl and CHX in the reduction of bacterial count (P>0.05) although the absolute mean bacterial count of NaOCl was lower than that of CHX. SEM results confirmed the microbiologic count results in both groups (EndoActivator and standard irrigation), there was no significant difference in the scores for remaining biofilm coverage between NaOCl and CHX at any of the 3 levels (P>0.05) although NaOCl was better than CHX in biofilm removal. Among the three scanned regions, coronal, middle and apical, there was a statistically significant difference between (NaOCl, CHX) and saline (P<0.05). Both were more effective at removing the biofilm with dentinal tubules free of bacteria than saline solution that was only partially effective at biofilm removal with persistent bacteria not only on the root canal wall but also within the dentinal tubules. There was no significant difference between scanned regions although it may have been anticipated that there would have been less effective biofilm removal apically and the better biofilm removal was at the coronal region. However, EndoActivator showed statistically non-significant low score rating for presence of E.faecalis biofilm on root dentin surface than standard syringe irrigation (P>0.05). Conclusions: The irrigate type rather than the device type is more effective in removal of biofilm and Both 5.25% NaOCl, 2% CHX, were effective in removal of Enterococcus faecalis biofilm.