A multidisciplinary investigation of the Eocene-Oligocene transition in the International Continental Scientific Drilling Program (ICDP)–U.S. Geological Survey (USGS) Eyreville core from the Chesapeake Bay impact basin was conducted in order to document environmental changes and sequence stratigraphic setting. Planktonic foraminifera and calcareous nannofossil biostratigraphy indicate that the Eyreville core includes an expanded upper Eocene (Biozones E15 to E16 and NP19/20 to NP21, respectively) and a condensed Oligocene-Miocene (NP24–NN1) sedimentary sequence. The Eocene-Oligocene contact corresponds to a ≥3-Ma-long hiatus. Eocene-Oligocene sedimentation is dominated by great diversity and varying amounts of detrital and authigenic minerals. Four sedimentary intervals are identified by lithology and mineral content: (1) A 30-m-thick, smectite- and illite-rich interval directly overlies the Exmore Formation, suggesting long-term reworking of impact debris within the Chesapeake Bay impact structure. (2) Subsequently, an increase in kaolinite content suggests erosion from soils developed during late Eocene warm and humid climate in agreement with data derived from other Atlantic sites. However, the kaolinite increase may also be explained by change to a predominant sediment input from outside the Chesapeake Bay impact structure caused by progradation of more proximal facies belts during the highstand systems tract of the late Eocene sequence E10. Spectral analysis based on gamma-ray and magnetic susceptibility logs suggests influence of 1.2 Ma low-amplitude oscillation of the obliquity period during the late Eocene. (3) During the latest Eocene (Biozones NP21 and E16), several lithological contacts (clay to clayey silt) occur concomitant with a prominent change in the mineralogical composition with illite as a major component: This lithological change starts close to the Biozone NP19/20-NP21 boundary and may correspond to sequence boundary E10–E11 as observed in other northwest Atlantic margin sections. It could result from a shift to more distal depositional environments and condensed sedimentation during maximum flooding, rather than reflecting a climatic change in the hinterland. The distinct 1‰ increase of the oxygen isotopes may correspond to the short-term latest Eocene “precursor isotope event.” (4) The abrupt increase of sediment grain-size, carbonate content, and abundance of authigenic minerals (glauconite) across the major unconformity that separates Eocene from Oligocene sediments in the Eyreville core reflects deposition in shallower settings associated with erosion, winnowing, and reworking. Sediments within the central crater were affected by the rapid eustatic sea-level changes associated with the greenhouse-icehouse transition, as well as by an abrupt major uplift event and possibly enhanced current activity on the northwestern Atlantic margin.