This research offers a comprehensive examination of calcareous nannofossils, isotopic ratios (δ18O and δ13C), and carbonate content spanning the critical Paleocene-Eocene transition at Gebel Nukhul. The study covers three lithostratigraphic formations: Tarawan, Esna, and Thebes. By meticulous analysis, we’ve identified five distinct calcareous nannofossil zones, including NP7/8 and NP9-12. Zone NP9 is further divided based on the lowest occurrences of specific nannofossils (Discoaster anartios, D. araneus, and/or Rhomboaster spp.), while Zone NP10 is subdivided using Tribrachiatus contortus as a marker. We discussed the significance of various calcareous nannofossil taxa in biostratigraphy, highlighting their role as valuable stratigraphic markers. Our research precisely locates the base of the Eocene at the onset of Subzone NP9b within the Esna Formation, revealing no discernible lithological changes coinciding with this boundary. Additionally, our findings align with the global record, indicating significant decreases in δ13C, δ18O, and calcium carbonate (CaCO3) content during the Paleocene–Eocene transition. These changes underscore the climatic and environmental perturbations during this pivotal geological phase. This study provides valuable insights into the stratigraphy and paleoclimatology of the Paleocene–Eocene transition, contributing to our understanding of past climatic variations on Earth.