Prehistoric dental treatments were extremely rare, and the few documented cases are known from the Neolithic, when the adoption of early farming culture caused an increase of carious lesions. Here we report the earliest evidence of dental caries intervention on a Late Upper Palaeolithic modern human specimen (Villabruna) from a burial in Northern Italy. Using Scanning Electron Microscopy we show the presence of striations deriving from the manipulation of a large occlusal carious cavity of the lower right third molar. The striations have a “V”-shaped transverse section and several parallel micro-scratches at their base, as typically displayed by cutmarks on teeth. Based on in vitro experimental replication and a complete functional reconstruction of the Villabruna dental arches, we confirm that the identified striations and the associated extensive enamel chipping on the mesial wall of the cavity were produced ante-mortem by pointed flint tools during scratching and levering activities. The Villabruna specimen is therefore the oldest known evidence of dental caries intervention, suggesting at least some knowledge of disease treatment well before the Neolithic. This study suggests that primitive forms of carious treatment in human evolution entail an adaptation of the well-known toothpicking for levering and scratching rather than drilling practices.