Biochronology is the most widely used method for organizing the successions of continental vertebrate faunas in geological time and for allowing the correlation of continental deposits. However, due to the fragmentary nature of the paleontological record and the diachronicity of first and last occurrences of vertebrate taxa in different areas (i.e., bioevents), it is difficult to precisely define the temporal boundaries between biochronological units. That is why it is crucial to calibrate biochronological data with independent proxies wherever possible. Here, thanks to an interdisciplinary approach that combines sedimentology and stratigraphy, vertebrate paleontology, micropaleontology (ostracods), palynology, and geochronology, we provide a chronological framework and a paleoenvironmental reconstruction of the paleontological site of Pantalla (Central Italy). The combination between biochronological/biostratigraphic and geochronological (U-series/ESR dating and paleomagnetism) data allows us to refer the site to c. 2.2 Ma, i.e., in the transition phase between the Middle and Late Villafranchian in the European biochronological scheme. We reconstruct the environment as a fluvial area with a frequently flooded wet floodplain and marked (probably seasonal) variations of the water level of the channel river. The depositional system was surrounded by a conifer-dominated forest, which is suggestive of a glacial phase.