Background and Aims . Retrieval of separated rotary file may not be possible many times. Then sealing of coronal root canal system depends on sealing ability of root canal sealers. The type of seal to be obtained should be bacterial-tight seal. Hence it is important to evaluate the sealing ability of obturation with different newer sealers with separated file in apical 3rd of root canals with bacterial penetration method using e.fecalis. Methods: Forty six human mandibular molars extracted for periodontal reasons were collected. After Decoronation of teeth Protaper rotary S1 files (Dentsply Maillefer) were intentionally separated in apical third of canal and obturated with gutta-percha (Prime Dental Products, India) along with zinc oxide eugenol sealer (Dental products of India-Mumbai) in Group I, Nano Zinc oxide eugenol (Nano Research Lab, Jharkand, India) in Group II and MTA sealer (Angelus, Londrina, Brazil) in Group III. Samples were assessed for bacterial leakage with E fecalis for 48 hours. The number of colony forming units was assessed. Results: The values obtained were subjected to one way ANOVA test and significance level was set to 0.05. Group I scored the highest mean value of (6.58), followed by Group II (3.91). The lowest Mean value was with Group III (2.91). However there was no statistical significant difference in Mean values among groups with (p-value 0.05). Conclusion: The mean value of colony forming units in Group III was 2.91, in Group II 3.91 and 6.58 in Group I. None of the groups demonstrated complete bacteria-tight seal.