Aim: The aim of this study was to compare the antimicrobial efficacy of calcium hypochlorite (Ca(OCl)2) and sodium hypochlorite (NaOCl) associated with or without erbium, chromium: yttrium, scandium, gallium, garnet (Er,Cr: YSGG) laser irradiation in root canals experimentally infected with Enterococcus faecalis. Materials and Methods: The root canals of 105 single-rooted premolars were enlarged up to file 40, autoclaved, inoculated with E. faecalis, and incubated for 21 days. The teeth were randomly divided into seven experimental (n = 15) groups according to the protocol for decontamination: syringe irrigation (SI) with distilled water (DW), SI with 2.5% NaOCl, SI with 2.5% Ca (OCl)2, laser-activated irrigation (LAI) with DW, LAI with 2.5% NaOCl and LAI with 2.5% Ca (OCl)2 and LAI with no solution. Microbiological samples were collected and the colony-forming units were counted before and after irrigation procedures and the percentages of reduction were calculated. Results: Mean numbers of recovered bacteria in the SI with DW group were significantly higher than those in the other experimental groups (P < 0.05). The high bactericidal reduction was observed in any of the other treatment groups: SI using NaOCl or Ca (OCl)2 and LAI with DW, NaOCl, Ca (OCl)2 or no solution, with no statistically significant difference. Conclusion: Er,Cr: YSGG laser with or without an irrirgation solution has antimicrobial effects on dentinal tubules infected with E. faecalis. The antimicrobial property of 2.5% Ca (OCl)2 was effective as 2.5% NaOCl on E. faecalis with conventional or laser activated irrigation in root canals.