Endodontic repair materials such as mineral trioxide aggregate (MTA) are used for various endodontic procedures. An alternative material to MTA with purportedly improved handling properties is EndoSequence Root Repair Material, which is available as premixed putty (ESP) or syringeable paste (ESS) and is described as possessing antibacterial activity during its setting reaction due to its highly alkaline pH. The aim of this in vitro study was to determine whether ESP and ESS possess antibacterial properties against a collection of Enterococcus faecalis strains recovered from root canal infections. The hypotheses tested were that (1) ESP and ESS possess antibacterial activity during their setting reaction, (2) there is no difference between ESP, ESS, and MTA in antibacterial activity, and (3) E. faecalis strains isolated from root canals differ in susceptibility to the materials. The direct contact test was used. ESP, ESS, and white MTA were preincubated at 37°C in >95% humidity for 30 minutes and 24 hours before 1-hour direct contact exposure to E. faecalis strains (n = 10). Absence of antibacterial carryover effect from the materials to the bacterial cultures was confirmed. Log(10) viable counts were compared by using analysis of variance with significance level at P ≤ .05. Combining data for all strains, the mean (± standard deviation) log(10) viable counts for ESP (4.55 ± 0.85), ESS (4.5 ± 0.95), and MTA (4.12 ± 1.26) were significantly lower than for untreated controls (7.40 ± 0.33) (P < .0001). The reduction in viable counts ranged from 1.86 ± 0.24 to 4.78 ± 0.42, with no statistically significant differences between the materials or preincubation periods. One strain was significantly more susceptible than 4 other strains. ESP, ESS, and MTA had similar antibacterial efficacy against clinical strains of E. faecalis. Clinical strains varied in their susceptibility to the root repair materials. Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.