The aim of this in vitro study was to evaluate the depth of effectiveness of erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser irradiation on microorganism reduction. From human roots, dentin slices of 100 microm to 1,000 microm thickness were prepared. These specimens were sterilized and then inoculated with 1 microl of Enterococcus faecalis suspension. The backs of the specimens were then irradiated with Er,Cr:YSGG radiation at a pulse energy of 3.13 mJ, delivered at an incidence angle of 5 degrees to the dentin slice surface. A control group was left without irradiation. The remaining bacteria were collected in 1 ml sterilized NaCl solution, serially diluted and seeded in Columbia-Agar plates. Despite the low pulse energy of 3.13 mJ, the Er,Cr:YSGG laser irradiation resulted in significant bacterial reduction up to a dentin thickness of 500 microm (P < 0.05). Scanning electron microscopy (SEM) micrographs of the contaminated and irradiated surfaces showed the absence of a smear layer and opened dentinal tubules.