We report a newly discovered hydrocarbon seep deposit from the Eocene bathyal flysch, exposed in the town of Buje in Istria, Croatia. Molecular fossils of methane‐oxidizing prokaryotes and abundant banded botryoidal cements indicate strong fluid flux at this site. We systematically describe the fauna of this and another seep deposit previously reported from Buje. The faunal assemblages are composed of eight species, these being an unidentified solemyid protobranch bivalve, the nuculid Nucula bowerbanki?, the nuculanid Nuculana? sp., the mytilid Brachidontes? amanoi sp. nov., the two thyasirids Channelaxinus dinaricus sp. nov. and Thyasira histriaensis sp. nov., the lucinid bivalve Amanocina bujensis sp. nov., and a possible provannid gastropod. The two assemblages are of low diversity (4 and 5 species, respectively), and are dominated by chemosymbiotic species whose occurrence is largely restricted to seeps. Despite their spatial and stratigraphic proximity, the two deposits share only a single species, Channelaxinus dinaricus, probably due to different fluid flux regimes at both seeps. The Buje seep assemblages are among the very few Late Cretaceous to Palaeogene chemosynthesis‐based faunal assemblages from the Tethys Ocean (the others being Late Cretaceous vent assemblages from Cyprus). From an evolutionary perspective, the Buje seep communities consist of genera with Mesozoic origins but lack Cenozoic novelties such as bathymodiolin mussels and vesicomyid clams, which are known from coeval deposits from the Pacific and dominate vents and seeps today. Thus, the Buje seep fauna support previous assertions that the Eocene Tethyan seep faunas preserved an ancient aspect, whereas evolutionary novelties arose in the Pacific.