The ancient harbour of Pisa, Portus Pisanus, was one of Italy’s most influential seaports for many centuries. Nonetheless, very little is known about its oldest harbour and the relationships between environmental evolution and the main stages of harbour history. The port complex that ensured Pisa’s position as an economic and maritime power progressively shifted westwards by coastal progradation, before the maritime port of Livorno was built in the late 16th century AD. The lost port is, however, described in the early 5th century AD as being “a large, naturally sheltered embayment” that hosted merchant vessels, suggesting an important maritime structure with significant artificial infrastructure to reach the city. Despite its importance, the geographical location of the harbour complex remains controversial and its environmental evolution is unclear. To fill this knowledge gap and furnish accurate palaeoenvironmental information on Portus Pisanus, we used bio- and geosciences. Based on stratigraphic data, the area’s relative sea-level history, and long-term environmental dynamics, we established that at ~200 BC, a naturally protected lagoon developed and hosted Portus Pisanus until the 5th century AD. The decline of the protected lagoon started at ~1350 AD and culminated ~1500 AD, after which time the basin was a coastal lake.