Purpose: This In vivo study was directed to compare and evaluate the bacterial count before and after preparing the root canals with the following variables: Single and multiple file rotary systems and Finishing after preparation with and without Xp-endo finisher. Methods: A total of 40 patient requiring endodontic treatment of both sexes were enrolled for this study from the dental hospital of Nahda University in BaniSuef. Patients were fully informed about the study`s procedures, benefits and risks. They signed an informed consent. Patients were divided into two groups according to the rotary system used ProTaper universal and one shape (n=20) and after preparation each group was subdivided into two subgroups according to the usage of XP-endo finisher or not (n=10). Irrigation during instrumentation performed with sterile saline. Three bacteriological samples were taken using paper points: S1: before mechanical preparation, S2: immediately after mechanical preparation, S3: 7 days after mechanical preparation before obturation. The plates were incubated for 24 hours at 37°c. The number of colony forming units (CFUs) was counted and calculated. Results: The bacterial count was reduced significantly in experimentation groups at S2 and S3 compared to S1. However, S3 compared to S2 show bacterial regrowth in the canal. For ProTaper groups there was a statistically significant difference between (XP) and (Without XP) groups. And also for OneShape groups there was a statistically significant difference between (XP) and (Without XP) groups, in the bacterial reduction.