Ancient Ostia at the mouth of the River Tiber into the Tyrrhenian Sea was largely significant for the economic supply of Rome. Ostia itself experienced an extraordinary period of prosperity in the second century AD. Starting in AD 42, a first new harbour at Portus was built by Emperor Claudius close to Ostia. It reached its full functionality under Emperor Trajan in the early second century AD, only. At Ostia itself, previous archaeological and geoarchaeological studies have brought to light a lagoon-type harbour at the western fringe of the city operating between the fourth and the second century BC in an artificially excavated harbour basin. From the second century BC onwards, a considerably smaller and shallower part of this western harbour basin was still in function as a fluvial harbour. So far, it was unclear whether Ostia’s western harbour was still in use when the harbour at Portus was set into function in the first to second century AD, or if the latter partially replaced Ostia’s harbour infrastructure. According to archaeological evidence, Ostia’s navalia-temple-complex, the main building at the eastern fringe of the western river harbour basin, was built in the second quarter of the first century AD. Was this prestigious harbour building erected although the associated harbour seemed to have been already given up before? We conducted detailed geoarchaeological investigations at the immediate western front of the navalia-temple complex. Results were compared with archaeological data obtained from excavations carried out in 2000/2001. A multi-proxy approach was used to reconstruct the history and evolution of the harbour. It was possible to identify subsurface structures and evaluate the local stratigraphy. Vibracoring brought to light a more than 1 m thick section of an opus reticulatum wall with parts of the original opus latericium on top. Such walls originally separated vaulted shipshed chambers of the navalia-temple complex at Ostia, which in turn formed the substructure of a temple complex located above it. Another core revealed the sedimentary infill of a former chamber of the building. Based on radiocarbon dating, the navalia was in use between the first and the fourth centuries AD with a water depth of maximum ca. 1.2 m at the immediate western front. This is in agreement with the date of construction of the navalia-temple complex in the second quarter of the first century AD. The relative sea level at that time was around 0.64 m below the present sea level. The harbour and the navalia were obviously accessible only for flat-keeled lighters and cargo boats. Larger cargo ships were either unloaded along the riverbank to the north of ancient Ostia (Hadler et al. 2019) or moored offshore, their freight being reloaded to smaller lighters. Chronostratigraphic data further show that the navalia-temple complex was in use until the second half of the fourth century AD. It was not before AD 355–363 or shortly afterwards, that the harbour site was abandoned. Ostia’s western river harbour was neither abandoned nor completely silted up before the harbour at Portus was established as previously assumed by other authors. Actually, the western front of the navalia-temple complex was hit by an extreme wave event, leaving a sand layer approx. 0.5 m thick, at or shortly after AD 355–363 which led to the final abandonment of Ostia’s western river harbour. This event is interpreted as a tsunami that may have hit the wider coastal region.