The organic-rich deposits of Late Devonian age are the second most important petroleum source rocks in the Ghadames Basin (Libya). From available stratigraphy, the peak of organic matter deposition, corresponding to a level of radioactive shales easily recognized in gamma ray logs, occurred at the Frasnian-Famennian transition. The present study was focused on samples from borehole D1–26, located in the central part of the Ghadames Basin, belonging to the Aouinet Ouenine III and IV formations (Frasnian to Famennian). The mineral and organic content of the rocks were analyzed in order to determine the origin of organic matter accumulation and estimate if the organic matter enrichment could be related to the well-known Upper Kellwasser event. The results indicate that during the Frasnian, the planktonic primary productivity was moderate, but anoxic conditions reaching the euphotic zone were frequent and allowed efficient preservation of the organic matter. At the Frasnian-Famennian transition, the deposition of the radioactive shales level can be related to a eustatic rise, which allowed incursion of nutrient-rich water in the basin, leading to increase in primary productivity and photic zone anoxia. During the Famennian, the conditions changed, associated with a relative sea level fall and/or a progradation of detrital sediments originating from the south. Photic zone anoxia was only episodic and plankton-derived organic matter was poorly preserved. Nevertheless, the important influx of organic matter of terrestrial origin allowed significant enrichment of the sediment in organic matter. Several indications suggest the radioactive shales interval could be coeval with the Upper Kellwasser level, nevertheless further stratigraphic work is needed to confirm this hypothesis.