The Eocene biota from the pre-rifting stage of the Upper Rhine Graben (southwest Germany) is, with exception of the world-famous fossil sites Messel and Bouxwiller, poorly known. While from these localities exclusively terrestrial and freshwater fossils were recovered, here we present floral and faunal assemblages from the middle Upper Rhine Graben which clearly indicate a temporarily brackish water environment and consist of a diverse palynomorph association, calcareous nannoplankton, foraminifers, ostracods and some fish and reptile remains. Based on the occurrence of Trochastrites hohnensis, a Lutetian age is assumed. Organic-walled dinoflagellate cysts, in particular Phthanoperidinium comatum, Phthanoperidinium echinatum, Apectodinium homomorphum and Apectodinium quinquelatum, suggest a late Ypresian to Lutetian age. The pollen and spores assemblage includes typical mid Eocene species, such as Tegumentisporis villosoides, Tricolporopollenites crassostriatus and representatives of Bombacaceae, but a late Ypresian to Priabonian age cannot be excluded. Foraminifers and ostracods do not further refine the biostratigraphical assignment. Hence, a Lutetian age is most probable. The mass occurrence of Neocyprideis, various foraminifer taxa and an organic-walled dinoflagellate cyst assemblage of very low diversity are indicative of a brackish water environment. Disarticulated vertebrate remains include fish teeth of Lepisosteidae, turtle plates and alligatoroid teeth of Diplocynodon and Hassiacosuchus. The present palaeogeographical scenarios do not consider a connection from the Upper Rhine Graben to the North Sea Basin, Alpine Sea/Paratethys or Paris Basin during the mid Eocene. Provided that the middle Upper Rhine Graben was land-locked and definitely not reached by a marine ingression during this time interval, we tentatively suggest that the brackish water taxa may have been accidentally introduced into a brackish inland sea by wind (anemochory), rain, highly mobile insects or vertebrates such as fish, birds and mammals (endozoochory/ectozoochory). The presumably freshwater calcareous nannoplankton species Nannoserratolithus minutus Martini is newly described.