The first detailed calcareous nannofossil and planktonic foraminiferal biostratigraphic and integrated lithofacies analyses of the Eocene–Oligocene transition at the Qa’ Faydat ad Dahikiya area in the Eastern Desert of Jordan, on the border with Saudi Arabia, is presented. Three calcareous nannofossil zones namely: Discoaster saipanensis (NP17), Chiasmolithus oamaruensis (NP18) and Ericsonia subdisticha (NP21), and three planktonic foraminiferal zones: upper part of Truncorotaloides rohri (E13), Globigerinatheka semiinvoluta (E14) and Cassigerinella chipolensis/ Pseudohastigerina micra (O1) are identified. Calcareous nannofossil bioevents recorded in the present study show numerous discrepancies with the Standard biostratigraphic zonal schemes to detect the Middle/Upper Eocene boundary (e.g. the highest occurrences (HOs) of Chiasmolithus solitus, C. grandis, and lowest occurrences (LOs) of C. oamaruensis, Isthmolithus recurvus are not considered reliable markers for global correlation). The Middle/Upper Eocene boundary occurs in the current study above the extinctions of large muricate planktonic foraminifera (large Acarinina and Truncorotaloides spp.) which coincide within the equivalent calcareous nannofossil NP18 Zone. These microplanktonic bioevents seem to constitute more reliable markers for the base of the Upper Eocene in different provinces. The uppermost portion of the Middle Eocene is characterized by an observed drop in faunal content and, most likely, primarily denotes the effect of the major fall in eustatic sea level. A major unconformity (disconformity) marked by a mineralized hardground representing a lowstand is recorded in the present study at the Eocene–Oligocene transition that reveals an unexpected ca. 2.1 Myr duration, separating Eocene (NP18/E14 zones) from Oligocene (NP21/O1 zones). Furthermore, the microfossil turnover associated with a rapid decline of the microfossil assemblages shows a distinct drop in diversity and abundance towards the Eocene/Oligocene unconformity and is associated with a sharp lithological break marked, at the base, by a mineralized hardground representing a major sequence boundary. These bioevents, depositional sequences and the depositional hiatus correlate well with different parts of the Arabian and African plates, but the magnitude of the faunal break differs from place to place as a result of intraplate deformation during the regional Oligocene regression of Neo-Tethys on the northern Arabian Plate. The presence of the Lower Oligocene shallow-marine calcareous planktonic assemblages in the study area indicate that communication between the eastern and western provinces of the western Neo-Tethys region still existed at this time.