This study aimed to evaluate the antibacterial action of KTP (potassium‐titanyl‐phosphate) laser irradiations (compared with 980 nm diode laser), associated with conventional endodontic procedures, on Enterococcus faecalis biofilms. Fifty‐six dental roots with single canals were prepared with Ni‐Ti rotary instruments, autoclaved, inoculated with an E. faecalis suspension and incubated for 72 h. They were randomly allocated to control and treatment groups. Laser parameters were as follows: power 2.5 W, Ton 35 ms, Toff 50 ms (KTP laser); power 2.5 W, Ton 30 ms, Toff 30 ms (980 nm diode laser). To evaluate the residual bacterial load, BioTimer Assay was employed. The chemo‐mechanical treatment together with laser irradiations (KTP and 980 nm diode lasers) achieved a considerable reduction of bacterial load (higher than 96% and 93%, respectively). Regarding both laser systems, comparisons with conventional endodontic procedures (mortality rate of about 67%) revealed statistically highly significant differences (P ≤ 0.01). This study confirms that laser systems can provide an additional aid in endodontic disinfection.