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Aim: The aim of this study was to compare the decrease in bacterial population in the root canals with 2.5% sodium hypochlorite (NaOCl) and three irrigation systems: Conventional Needle Irrigation, Passive Ultrasonic Irrigation, and EndoVac Apical Negative Pressure Irrigation systemMaterials and Methods: Access open was carried out in 48 extracted single-rooted maxillary and mandibular human premolar teeth. The canals were instrumented 1 mm short of apical foramen up to No. 50 size file; irrigated and apical foramen was sealed with cyanoacrylate. The teeth were mounted in plaster and autoclaved for 20 min at 121°C. The plaster blocks containing the teeth were inoculated with Enterococcus faecalis and incubated at 37°C for 24 h. The specimens divided randomly into four groups (n = 12) depending on the type of irrigation system used: Group 1: Conventional Irrigation Needle, Group 2: Ultrasonic system, Group 3: EndoVac Negative Pressure Irrigation system used with 2.5% NaOCl, and Group 4: Control group, normal saline irrigation. The normal saline is used as control. The samples were collected using 45 size sterile paper points and quantitative bacterial assessment was carried out by seeding each dilution on plates containing brain heart infusion broth. Then, incubated at 37°C for 7 days and colony-forming units were counted. Data obtained were statistically analyzed. Results: Statistically, EndoVac irrigation systems were more effective in reducing the number of bacteria than the other irrigation systems. Conclusion: EndoVac irrigation system was more effective in reducing bacterial numbers in infected root canals than the other two irrigation systems.