The aim of this study was to evaluate and compare the morphological changes that occur in dentine after femtosecond laser irradiation and after mechanical treatment. The duration of the laser pulse is an important parameter, because within the time frame of the pulse heat diffusion plays a very important role in the mechanism of interaction between the light and the tissue. Six totally impacted human third molars were sectioned into sheets approximately 1 mm thick with an Accutom-50 precision cutting machine. The samples were randomly divided into two groups according to their cavity preparation: mechanical cavity preparation and laser cavity preparation. The samples were then examined by light microscopy and scanning electron microscopy. There were clear differences in the results obtained with the two techniques. Cavities prepared with the laser with pulses of <1 ps showed no microcracks, and the treated surface displayed a rough and irregular aspect with no smear layer and exhibited open dentinal tubules. On the contrary, cavities made with a rotatory instrument had a smooth surface and microcracks, a broad area of carbonization and merging, occluded dentinal tubules and a smear layer. This study showed that human dentine can be successfully ablated with the ultrashort pulse laser.