Objectives Early evidence for the treatment of dental pathology is found primarily among food‐producing societies associated with high levels of oral pathology. However, some Late Pleistocene hunter‐gatherers show extensive oral pathology, suggesting that experimentation with therapeutic dental interventions may have greater antiquity. Here, we report the second earliest probable evidence for dentistry in a Late Upper Paleolithic hunter‐gatherer recovered from Riparo Fredian (Tuscany, Italy). Materials and Methods The Fredian 5 human consists of an associated maxillary anterior dentition with antemortem exposure of both upper first incisor (I1) pulp chambers. The pulp chambers present probable antemortem modifications that warrant in‐depth analyses and direct dating. Scanning electron microscopy, microCT and residue analyses were used to investigate the purported modifications of external and internal surfaces of each I1. Results The direct date places Fredian 5 between 13,000 and 12,740 calendar years ago. Both pulp chambers were circumferentially enlarged prior to the death of this individual. Occlusal dentine flaking on the margin of the cavities and striations on their internal aspects suggest anthropic manipulation. Residue analyses revealed a conglomerate of bitumen, vegetal fibers, and probable hairs adherent to the internal walls of the cavities. Discussion The results are consistent with tool‐assisted manipulation to remove necrotic or infected pulp in vivo and the subsequent use of a composite, organic filling. Fredian 5 confirms the practice of dentistry—specifically, a pathology‐induced intervention—among Late Pleistocene hunter‐gatherers. As such, it appears that fundamental perceptions of biomedical knowledge and practice were in place long before the socioeconomic changes associated with the transition to food production in the Neolithic.