The aim of this study was to evaluate the antibacterial activity of 2% iodine potassium iodide (IKI) used as a final rinse after the cleaning and shaping procedures in mesial root canals of mandibular molars infected with Enterococcus faecalis. Seventy two mandibular first molars were used. The root canals were infected with Enterococcus faecalisfor 30 days. After the infection procedures, the root canals were cleaned and shaped by using the Pro Taper rotary system and manual files. The teeth were randomly assigned to four experimental groups (N = 18). In group 1, the root canals were irrigated with sterile distilled water (control). In group 2, the root canals were irrigated with 1% Sodium hypochlorite (NaOCl) during instrumentation. In group 3, root canals were irrigated with 1% NaOCl during instrumentation and a five minute final irrigation using 2% IKI. In group 4, the root canals were irrigated with 1% NaOCl during instrumentation and a 15 minutes final irrigation with 2% IKI. Bacteria colony-forming units (CFU) from root canals were semi-quantified and the presence of negative cultures among the groups was compared using Fisher's test (p < 0.05). The order of effectiveness was: 1% NaOCI plus 2% IKl for 15 minutes (95%), 1% NaOCl plus 2% IKl for 5 minutes (44%), 1% NaOCl (17%) and sterile distilled water (0%). Fisher's exact test showed a significant difference among the groups (p < 0.05). It was concluded that under in vitro conditions, IKI was able to eliminate the Enterococcus faecalis from infected dentin significantly in a 15-minute time frame after the cleaning and shaping procedures.