Cranial trepanation is among the oldest head surgical procedures, creating a hole through the skull. This procedure is the surgical removal of a portion of the skull as a medical treatment to cure a specific disease or as a religious or magical ritual. Cranial trepanation has been reported from very early times and widespread all over the world. This work is based on data from six trepanned skulls from the Hellenistic Age (IV century BC) to the Late Antiquity (VI century). The samples come from Puglia (Southern Italy), from the northern (the peninsula of Gargano) to the central area: three cases come from Vieste (Foggia), Ischitella (Foggia) and Alberona (Foggia), the others three from Gravina in Puglia (Bari) and Canosa (Barletta, Andria, Trani). All skulls have been evaluated via an anthropological, radiological and odontological approach. This study indicates the value of a methodology, which encompasses anthropology, radiology and imaging, and archeo-forensic investigations, highlighting the need for multidisciplinary teamwork in any assessment of human remains. The study supports the hypothesis of the presence of a long tradition of medical centres in the Mediterranean basin area.