The Middle Late Eocene relatively poorly known from the southwestern Neo-Tethys settings since no integrated study is presented so far. The Souar section (604 m, 36°13'27.07N, 10° 1'23.77E) from the Tunisian dorsale (Zaghouan, Tunisia) provide the needed stratigraphic, chronological, and paleoenvironmental constraints for a reference section in the southern Neo-Tethys. We generate new high-resolution biostratigraphic, magnetic susceptibility, stable isotope and X-ray fluorescence (XRF) records spanning the Lutetian to lower Priabonian (~36.7 to 45.8 Ma). Quantitative calcareous microfossil has been performed (94 samples, ~6 m resolution) to identify the major biostratigraphic horizons (Base, base common, top, and top common) following the work of Agnini et al., 2014. Calcareous nannofossils zones extends from CNE9 to CNE18. The high-quality XRF and XRD records highlight the potential enhanced biosiliceous productivity during the lower Bartonian. This indicates the development of upwelling zone causing the observed drastic reduce of in situ calcareous nannoplankton and carbonate productivity. A correlation between the δ13Corg of the Souar section and other published reference sections (Pacific and Neo-Tethys) suggests the identification of the δ13C pre-MECO local maximum at ~40.5 Ma, of the δ13C MECO minimum at ~40 Ma, as well as a potential δ13C maximum at ~37.4 Ma. The Bartonian-Priabonian boundary is placed at the base of Reticulofenestra erbae (37.89 Ma) and coincides with major variations in calcareous nannofossil abundance, calcium carbonate content, and magnetic susceptibility indicating profound changes in hemipelagic carbonate production and the source of siliciclastic material, likely related to the late Eocene climate change and palaeoceanographic restriction of the Neo-Tethys.