To evaluate the antibacterial efficacy of HybenX, an endodontic irrigant against Enterococcus faecalis biofilm and determine its efficacy in removal of smear layer. Forty root canals were inoculated with E. faecalis for three weeks, divided into 4 groups and irrigated with: Group1: 5 ml 2.5% sodium hypochlorite; Group2: 5 ml 2% chlorhexidine gluconate; Group3: 5 ml HybenX; Group4: 5 ml distilled water (control). Dentin shavings were collected and plated on agar plates, followed by colony forming unit (CFU) determination. For smear layer removal examination, 30 single rooted teeth were instrumented and divided into 3 groups and treated with: GroupI: 5 ml of 17% EDTA; GroupII:5 ml of HybenX; and GroupIII: 5 ml of distilled water. Samples were then subjected to SEM examination. All irrigants showed a significant reduction in CFUs compared to the control group (P⟨ .001), but none compared to each other. Regarding smear layer removal in the apical third, EDTA removed smear layer more effectively than HybenX (P = .014). HybenX demonstrated good antimicrobial efficacy against E. faecalis biofilm and removed the smear layer effectively in coronal and middle third of the root canal system. HybenX can be considered as a promising irrigating agent in root canal treatment of infected teeth.