Three isolated limestone deposits and their fauna are described from a middle Eocene Flysch succession in northwestern Istria, Croatia. The limestones are identified as ancient methane-seep deposits based on fabrics and characteristic mineral phases, δ13Ccarbonate values as low as −42.2 ‰ and 13C-depleted lipid biomarkers indicative of methane-oxidizing archaea. The faint bedding of the largest seep deposit, the great dominance of authigenic micrite over early diagenetic fibrous cement, as well as biomarker patterns indicate that seepage was diffusive rather than advective. Apart from methanotrophic archaea, aerobic methanotrophic bacteria were present at the Eocene seeps as revealed by 13C-depleted lanostanes and hopanoids. The observed corrosion surfaces in the limestones probably reflect carbonate dissolution caused by aerobic methanotrophy. The macrofauna consists mainly of chemosymbiotic bivalves such as solemyids (Acharax), thyasirids (Thyasira) and lucinids (Amanocina). The middle Eocene marks the rise of the modern seep fauna, but so far the fossil record of seeps of this age is restricted to the North Pacific region. The taxa found at Buje originated during the Cretaceous Period, whereas taxa typical of the modern seep fauna such as bathymodiolin mussels and vesicomyid clams are absent. Although this is only a first palaeontological glimpse into the biogeography during the rise of the modern seep fauna, it agrees with biogeographic investigations based on the modern vent fauna indicating that the dominant taxa of the modern seep fauna first appeared in the Pacific Ocean.