Several studies have been conducted in order to verify effects of laser irradiation on dental structures. However, different settings and methods employed have led to inconclusive results. The aim of this study was to evaluate the effects of several settings of Nd:YAG laser irradiation on enamel microhardness. Fifteen groups of 10 human third molars each were submitted to Vickers microhardness indentations prior to and after laser irradiation. The data were submitted to the t-test for dependent samples (P < 0.05) for statistical evaluation. Lower total delivered energy (under 30 J) resulted in no statistically significant difference of microhardness between irradiated and non-irradiated samples, whereas higher energy settings decreased enamel surface microhardness significantly. This can be due to the significant temperature rise during irradiation. Thus, we conclude that laser irradiation, under the conditions of this study, is not suitable for improving enamel properties, because of decreased microhardness of irradiated samples.