Thirty-six bituminous samples from the Pre-Pottery Neolithic site of Nahal Efe (8000–7700 cal. BCE, Negev, Israel) were analysed to determine their origins. Most samples are mixtures of bitumen with minerals, predominantly quartz, calcite, dolomite and aragonite. The aragonite is from marine shells, which were crushed to be incorporated into the mixtures.Dead Sea bitumen was identified in all samples. The Nahal Efe geochemical data were compared to samples from Tell Yarmuth (2800–1100 BCE), Tall Dhiban (700–800 cal. BCE), Tel Gezer (1250–700 cal. BCE) and to floating asphalt blocks from the Dead Sea. The stable carbon isotope values of resins and asphaltenes of the Nahal Efe bitumens differ from those of the Dead Sea floating blocks, Tell Yarmuth, Tel Gezer and Tall Dhiban, apparently a consequence of diagenesis. Thus, δ13C values of asphaltenes may not always be a reliable representative of the stable carbon isotope composition of the unaltered source bitumen. Surprisingly, the sterane and terpane patterns of the Nahal Efe samples are not severely biodegraded and clearly indicate a Dead Sea bitumen source. No difference was recorded between mixtures and pure bitumen. Some changes have, however, been observed when using quantitative data on biomarkers. The shifts in δ13C values of resins and asphaltenes is not related to biodegradation but rather to oxidation of the Nahal Efe samples.This is the earliest and most complete evidence of Dead Sea bitumen exploitation and use, which included the preparation of different ‘recipes’, in the Negev and Sinai arid regions.