The purpose of this study was to compare the compositional changes and microhardness of the cavity floor prepared by erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser irradiation with those of the conventional bur-prepared cavity. A total of 16 extracted human molar teeth (with no carious lesions or repairs) were selected for this study. On the buccal and lingual (palatal) surfaces of each tooth, cavities (diameter 3 mm, depth 2 mm) were prepared with an Er,Cr:YSGG laser system (Waterlase MD(TM), USA) and high-speed turbine. The cavities were cross-sectioned and subjected to atomic analysis by scanning electron microscopy-energy dispersive X-ray spectrometry (SEM-EDX) and Vickers hardness test. Statistical analyses were performed with t-tests. Surface characteristics of the prepared cavities were also investigated by SEM. No significant differences were found between the calcium/phosphorus (Ca/P) ratio and Vickers hardness of laser- and bur-prepared cavities. The SEM observation revealed that the lased cavity surface was irregular, and there was also an absence of smear layer; the orifices of dentinal tubules were exposed. Microhardness measurement of the cavity floor confirmed that the Er,Cr:YSGG laser produced a clean-cut surface of the cavity.