The purpose of the study was to evaluate the effectiveness of different root canal disinfection techniques in the elimination of a multi-species biofilm from inside the root canal. Fifty mandibular first premolars were used in the present study, standardized to 11mm of root length, and instrumented with a reciprocation system Reciproc, (VDW GmbH, Munich, Germany) to a #50. Longitudinally sectioned halves of the roots were obtained and washed with NaOCl 4%, EDTA 17% and 5% sodium thiosulfate, and sterilized by autoclaving for 15 minutes at 121°C. A multi-species biofilm broth was developed with three strains of bacteria under laboratory conditions: Enterococcus faecalis ATTC 29212, Eikenella corrodens ATTC 23834, Streptococcus anginosus ATTC 33397. Roots were autoclaved and transferred to the broth for 4 days and then were subjected to either disinfection with sodium hypochlorite 4% and XP-endo Finisher (FKG Dentaire, La Chaux-de-Fonds, Switzerland) or chlorhexidine 2% with and without activation with XP-endo Finisher (FKG Dentaire, La Chaux-de-Fonds, Switzerland). The evaluations of the biofilm elimination showed results that indicate that the 4% sodium hypochlorite group with positive pressure irrigation presented significant differences with the group that had irrigation with sodium hypochlorite activated with XP-endo Finisher and the chlorhexidine groups to 2% (P<0.05). Chlorhexidine 2% activated with the XP-endo Finisher does not exert elimination or improved cleaning effect on the multi-species biofilm. Activation of sodium hypochlorite 4% improved the elimination of the multi-species biofilm.