Objective: The purpose of this in vitro study was to investigate the ability of Er,Cr:YSGG laser irradiation at different energy settings to eliminate Candida albicans, either with or without 5.25% sodium hypochlorite (NaOCl) treatment. Methods: In total, 90 extracted human single-rooted teeth were shaped by using a crown-down technique with HERO Shaper rotary instruments and irrigated with 1 mL of 2.5% NaOCl between each instrument. The specimens were autoclaved and incubated with a suspension of C. albicans (ATCC 90028). Then the specimens were divided into six treatment groups (n 15 per group): group 1, NaOCl+1-W laser; group 2, 1-W laser; group 3, NaOCl+0.75-W laser; group 4, 0.75-W laser, group 5, NaOCl; and group 6, no treatment. Aliquots of the experimental teeth were transferred onto Sabouraud's dextrose agar plates with paper points, and colony-forming units (CFUs) were counted as a measure of antifungal activity. One root from each group, which was not submitted to the sampling procedure, was fixed in glutaraldehyde, split into two halves, and evaluated with scanning electron microscopy (SEM). Statistical analysis was performed by using analysis of variance (ANOVA) and the Dunnett t test. Significance was set at alpha-0.05. Results: All the treatment groups resulted in a significant reduction of C. albicans, but no sterilization, and all the groups were significantly different from each other (p < 0.0001). Group 1 (NaOCl and 1-W laser combination) exhibited the greatest reduction, whereas group 5 (NaOCl) resulted in a minimal reduction in CFUs of C. albicans. The SEM observations were in accordance with the microbiologic analysis. Conclusions: The 5.25% NaOCl and 1-W laser treatment combination may be a valuable cleaning tool for root canals infected with C. albicans.