This study determined the compositional changes and microhardness of the cavity floor prepared by Er,Cr:YSGG and Er:YAG lasers and compared the results with the conventional method of bur preparation. Fifteen non-carious human molars were used in this study. On the buccal and lingual surfaces of each tooth, two cavities (mesio-distal 3 mm, inciso-gingival 3 mm, depth 2 mm) were prepared with two different laser devices (Er,Cr:YSGG laser; Waterlase MD and Er:YAG laser; KaVo Key Laser 3) and a high-speed turbine. The teeth were embedded into polyester resin and cross-sectioned. The microhardness measurements from the floor of each half cavity were recorded with the Vickers surface hardness tester. The remaining halves of the cavities were subjected to SEM-EDS atomic analysis. The results were statistically evaluated by one-way ANOVA and Kruskal Wallis tests (p = 0.05). No significant differences were observed among the microhardness values, quantities of Ca (Ca weight %), P (P weight %) and Ca/P ratio of the lased and conventionally prepared cavities (p > 0.05). The cavity preparation techniques and differences in laser devices did not significantly alter the composition and microhardness of dentin tissue. Both laser devices used in this study were observed to lead to minimal thermal damage in the dentin tissue and minimal thermally-induced changes in dentinal compositions.