Theopetra Cave is a unique prehistoric site for Greece, as the Middle and Upper Paleolithic, Mesolithic, and Neolithic periods are present here, bridging the Pleistocene with the Holocene. During the more than 20 yr of excavation campaigns, charcoal samples from hearths suitable for 14C dating were collected from all anthropogenic layers, including the Paleolithic ones. Most of the samples were initially dated using the ABA chemical pretreatment protocol in the Laboratory of Archaeometry of NCSR Demokritos, Greece, and the Radiocarbon Dating and Cosmogenic Isotopes Laboratory of the Weizmann Institute of Science, Israel. The 14C results, which were not always consistent versus depth, showed that the earliest limit of human presence is ∼50,000 yr BP, thus reaching the age limits of the 14C dating method. However, 10 TL-dated burnt flint specimens unearthed from the lower part of the Middle Paleolithic sequence of the cave gave ages ranging between ∼110 and 135 kyr ago. These results are in disagreement with the 14C dates, as they support a much later date for these layers. In order to clarify the situation further, charcoal samples originating from hearths were conventionally dated in the Laboratory of Archaeometry of NCSR Demokritos using the ABA pretreatment. Additionally, hand-picked charcoal fragments also underwent 14C dating by AMS in the Oxford Radiocarbon Accelerator Unit using the acid-base wet oxidation (ABOX-SC) pretreatment protocol. The 14C dates from the cave's Paleolithic layers obtained by both pretreatment protocols suggest a probable charcoal diagenesis affecting the 14C results of these very old samples. However, the dates obtained with ABOX-SC pretreatment are considered more reliable and in the younger stratigraphic part produced consistent results with the TL dating.