This study aimed to evaluate the influence of needle design and irrigant flow rate on the removal of Enterococcus faecalis mature biofilms during sodium hypochlorite irrigation. Forty-eight single-rooted human teeth were instrumented (ProTaper F3), autoclaved and inoculated with Enterococcus faecalis to establish a two-week-old biofilm. E. faecalis biofilms were treated with Sodium hypochlorite that was injected in the root canals using three types of needles (NaviTip, ProRinse, IrriFlex). For the IrriFlex needle, one, two, or four bars of pressure was applied to the irrigating solution to increase flow rates. Bacteria were labeled with the LIVE/DEAD BacLight Bacterial Viability kit, and viability was assessed by flow cytometry (FCM). Results were statistically analyzed using one-way ANOVA and Tukey multiple comparison intervals (α = 0.05). Bacterial viability was significantly reduced after sodium hypochlorite passive irrigation but the number of viable bacteria retrieved from root canal specimens irrigated with the Pro-Rinse needle was significantly higher compared to NaviTip and IrriFlex needles (p < 0.05). When the irrigant flow rate was increased, the viability of bacterial biofilms was significantly reduced compared to passive irrigation using the IrriFlex needle (p < 0.05). Applying higher flow rates during irrigation using the IrriFlex needle did not further reduce bacterial viability.