On the Island of Samos (East Aegean region, Greece), two sedimentary basins are filled by thick continental series dated to the Late Miocene to Early Pliocene. A multi‐disciplinary study has been performed including 1) the definition of 21 sedimentary facies, 2) a review of the biological components and 3) carbon, oxygen and strontium stable isotope analyses. The succession is characterised by various depositional settings and hydrochemical compositions. Five main stages of basin evolution have been identified: 1) The Late Serravalian is marked by the development of alluvial fans and fan delta; 2) during the Lower Tortonian, isolated shallow lakes with variable salinity, from fresh to brackish, developed under warm and relatively humid conditions; 3) the Middle to Upper Tortonian is marked by the development of a large and deep lake with saline and alkaline waters, under colder and drier conditions; 4) the Latest Tortonian to Messinian period is represented by an ephemeral alluvial system, developed under a dry climate; 5) during the Zanclean, a palustrine and paludal wetland system, dominated by tufa carbonates, developed under moderately humid conditions. This succession is of particular interest for the reconstruction of the palaeoenvironmental evolution of the transition zone between the Mediterranean domain, and the Paratethys and circum‐Paratethys areas. The geochemical data and the presence of flora (diatoms) and fauna (gastropods) of marine affinity suggest transient ingressions of marine‐related water or groundwater inflows as early as the Lower Tortonian. The Samos succession records the complex interaction between the regional geodynamics and climate. The extensional regime of the Eastern Aegean zone generates subsidence, interrupted in the mid‐Tortonian (9 Ma) by a brief compressive event and a major exposure of the basins. Furthermore, the Late Miocene progressive aridification, followed by a change to a more humid climate (Pliocene) is also a major driver of the sedimentation.