Abundant and well-preserved diatoms and silicofl agellate assemblages are documented through a complete late Eocene sequence, ODP Hole 1090B, recovered from the southern Agulhas Ridge in the sub-Antarctic South Atlantic. A sequence of Cestodiscus (diatom) species occurrence events involving C. pulchellus var. novazealandica, C. fennerae, C. antarcticus, C. convexus, C. trochus, and C. robustus is tied with paleomagnetic stratigraphy and provides the basis of proposing a new diatom zonation for the latest middle Eocene to early Oligocene (~37.6–33.4 Ma) of the sub-Antarctic South Atlantic. Comparison with previously published diatom occurrence charts suggested this zonation should be applicable throughout the low latitude regions of the world’s oceans. Silicofl agellates belong to the Dictyocha hexacantha and the overlying Corbisema apiculata Zones. The late Eocene succession of silicofl agellate species is dominated by Naviculopsis (20–60%). Naviculopsis constricta and N. foliacea dominate the D. hexacantha Zone, followed by the N. constricta, then N. biapiculata in the C. apiculata Zone. Cold-water Distephanus is most abundant in the latest Eocene along with N. biapiculata. The tops of zonal guide fossils Dictyocha hexacantha and Hannaites quadria (both 36.6 Ma) and Dictyocha spinosa (37.1 Ma) are tied with paleomagnetic stratigraphy.