The aim of this study was to compare the efficacy of root canal irrigants against E. faecalis biofilms using a novel in vitro testing system. Biofilms grown in a flow cell system were submerged in test irrigants for either 1 or 5 minutes. Statistical analysis revealed a significant relationship between test agent and percentage kill of the biofilm bacteria (P < 0.05). No statistically significant relationship between time and percentage kill was found. The percentage kill of the biofilm bacteria was: 6% NaOCl (>99.99%), 1% NaOCl (99.78%), Smear Clear (78.06%), 2% chlorhexidine (60.49%), REDTA (26.99%), and BioPure MTAD (16.08%). Post-hoc analysis showed a significant difference between 1% and 6% NaOCl, and all other agents including Smear Clear, 2% chlorhexidine, REDTA, and BioPure MTAD (P < 0.05). Within the parameters of this study, both 1% NaOCl and 6% NaOCl were more efficient in eliminating E. faecalis biofilm than the other solutions tested.