Within the Neogene-Quaternary evolution of the Mediterranean intermountain basins, the eastern Tiberino Basin provides new multifaceted chronological, biostratigraphic, palaeoecological, and palaeoenvironmental information, appreciably improving the knowledge of palaeoenvironmental and climate conditions during the middle-late Matuyama Chron (late early Pleistocene). Shallow to relatively deep lacustrine deposits and alluvial plain deposits, magnetostratigraphically calibrated, hold malacofaunas, ostracofaunas, and carpological remains, as well as a pollen record. Palaeocarpological remains widely originated from the local (azonal) vegetation of waterlogged environments. Nonetheless, some taxa show transitional morphology between possibly extinct Pliocene-Pleistocene forms and living taxa. The pollen record highlights a conifer-dominated forest phase, indicating a temperate-wet interglacial period, well aligned inside the schemes for the same latitudinal band. The abundance of tree taxa currently absent from the Italian peninsula points to pre-Jaramillo late early Pleistocene biostratigraphical characters, here compared to other sections from central Italy, and contributes to a better definition of modes and timing of their disappearance in southern Europe. Malacofaunas and ostracods, still with late early Pleistocene features, together with Charophyte, mark repeated fluctuations in energy, temperature, and chemical composition of water. The overall record identifies an incipient diachronous cooling trend, for the first time recognized in southern Europe.