This in vitro study was mainly aimed to evaluate the effect of high-frequency alternating currents (AC) applied by an electronic apex locator (EAL) on the antibacterial properties of chlorhexidine (CHX) on E. faecalis biofilm. The root canals of 120 extracted human single-rooted teeth were prepared using Gates-Glidden drills and hand K-files. After contaminating the root canals with E. faecalis, they were incubated for 60 days. Then, the teeth were randomly divided into six experimental groups (n=20). Group 1, 2% CHX; group 2, normal saline (NS) with direct current (DC); group 3, normal saline (NS) with high-frequency alternating current (AC); group 4, 2% CHX with DC; group 5, 2% CHX with AC; group 6, control (normal saline). The samples were collected from the root canal walls of 16 teeth in each group and 1:10 serial dilutions were prepared and added to Muller-Hinton agar (MHA) plates and incubated at 37°C for 48 h. The longitudinal sections of the other 4 teeth used to observe under a scanning electron microscope (SEM). A classic colony counting technique was used for counting the vital E. faecalis bacteria in MHA. Two-way ANOVA was used for statistical analysis of the data. The level of significance was set at P<0.05. The electric current significantly changed the colony-forming units (CFU) values (P<0.001). According to pair-wise comparisons, the highest CFU difference was observed between the AC group and the group without electric current (P<0.001); furthermore, the difference between the DC group and the group without electric current was not significant (P=0.823). The highest bioelectric effect occurred with the use of high-frequency alternating electric current in the form of an apex locator with CHX as a canal irrigant.