Non surgical endodontic therapy uses biomechanical cleansing and shaping of the canal, which may be combined with the application of antimicrobial irrigants and intracanal medications, to eliminate these bacteria. The study evaluated the antimicrobial efficacy of Ocetenidine Dihydrochloride, a newer irrigant with Sodium Hypochlorite, a gold standard against Enterococcus faecalis in root canal disinfection. 30 single rooted intact pre-molar teeth were collected. The teeth were decoronated to measure 7mm long segments. The root apexs were enlarged to size 40 using K files. The root surface were coated with nail varnish and subsequently the root apex was sealed using light cure composite resin. Following sterilization of the blocks, they were infected with pure cultures of E.faecalis, which was grown in Muller-Hinton agar for a period of 7 days. The specimens were divided into 3 distinctive groups containing 10 teeth each: Group I with Octenidine, (0.1%),Group II with NaOCL (5%),Group III with no irrigation (control). Each block was carefully removed and was irrigated with 3ml of the irrigant for 1 min. After irrigation, the dentine debris were harvested from the dentine blocks using Gates Glidden drills No.2 and No.3. The antibacterial assessment was carried out by finding out the MIC and MBC values by serial dilution method, and the proportions of viable bacteria under epifluorescence microscope using Syto9 dye. Statistical analysis comparing the inter-group was done using MANN-WHITNEY U test and KRUSKAL-WALLIS test. From the results it was observed that the antimicrobial efficacy of Octenidine was found to be higher than 5% Sodium hypochlorite against E.faecalis and in creating a “bacteria-free” root canal system.