The territory has been inhabited since the Palaeolithic, and its caves sheltered the first communities. The most remarkable find is Delia , the "Ostuni woman" — the skeleton of a young woman in an advanced state of pregnancy, recovered from the cave of Santa Maria di Agnano , and dated to somewhere between twenty-five and twenty-eight thousand years ago ; the estimates differ. Neolithic agricultural finds have come from Lamaforca and San Biagio .

The first organised town was Messapian . That Illyrian people, able builders of roads and cities, settled the Salento in the first millennium BC — sources give both around 1000 BC and the seventh century — and Ostuni's first urban nucleus is theirs. In the third century BC it was absorbed into Rome with the rest of the Salento, though little Roman architecture survives.

Under the Aragonese the town flourished as an agricultural and commercial centre, and the walls that ring the old town are Aragonese work.

The cathedral is the exception that proves a rule about Apulian architecture. Begun in 1435 and completed by 1495 , it is late Gothic — rare in a region overwhelmingly Romanesque or Baroque. It sits on the summit of the highest hill, and its facade is tripartite, divided by pilasters: the central section ends in a tympanum formed of two ogee arches, the wings in half-lunettes, and a Gothic crowning of trilobed arches on carved corbels runs along the flanks and around the transept. Three ogival portals open below — the central lunette carries a relief of the Madonna and Child in glory — each surmounted by a rose window. The central rose has twenty-four rays and is reckoned among the largest and finest of its kind in Europe; Christ stands at its centre, surrounded by columns representing the hours of the day. Inside, the Latin-cross plan on three naves was restored so often that the eighteenth century effectively remade it, and the flat ceiling carries canvases of that century.

Opposite stand the Palazzo Vescovile and the old Seminario , joined by a finely worked stone arch that bridges the street between them.

The seventeenth century brought plague, and with it the devotion that still defines the town. Sant'Oronzo is credited by tradition with delivering Ostuni from the disease, as he is in other cities of the Salento. It is very probably the same period that fixed the whitewashing: lime was applied against contagion, and what began as public health became the town's identity.

In 1771 the Baroque obelisk of Sant'Oronzo was carved and raised in the main square — nearly twenty-one metres high — in thanks for the saint's protection against cholera and earthquakes.

Ostuni also has a Risorgimento record its size would not predict. Its citizens founded a circle of the Giovine Italia and a Carbonari outlet, and during the risings Ostuni was the first city in Puglia to raise the tricolour .

From the 1930s it became the principal tourist centre of what was by then the province of Brindisi.