In 1513, the Portuguese were the first Europeans to land in Ambon, and it became the new centre for Portuguese activities in Maluku following their expulsion from Ternate. The Portuguese, however, were regularly attacked from native Muslims on the island's northern coast, in particular Hitu, which had trading and religious links with major port cities on Java's north coast.
They established a factory in 1521, but did not obtain peaceable possession of it until 1580. Indeed, the Portuguese never managed to control the local trade in spices, and failed in attempts to establish their authority over the Banda Islands, the nearby centre of nutmeg production.
The Portuguese were dispossessed by the Dutch already in 1605, when Steven van der Hagen took over the fort and without a single shot. Ambon was the headquarters of the Dutch East India Company (VOC) from 1610 to 1619 until the founding of Batavia (now Jakarta) by the Dutch.
About 1615 the English formed a settlement on the island at Cambello, which they retained until 1623, when it was destroyed by the Dutch. Frightful tortures inflicted on its unfortunate inhabitants were connected with its destruction. In 1654, after many fruitless negotiations, Oliver Cromwell compelled the United Provinces to give the sum of 300,000 gulden, as compensation to the descendants of those who suffered in the "Ambon Massacre", together with Manhattan.
In 1673 the poet John Dryden produced his tragedy Amboyna; or the Cruelties of the Dutch to the English Merchants. In 1796 the British, under Admiral Rainier, captured Ambon, but restored it to the Dutch at the peace of Amiens, in 1802. It was retaken by the British in 1810, but once more restored to the Dutch in 1814. Ambon used to be the world center of clove production; until the nineteenth century, the Dutch prohibited the rearing of the clove-tree in all the other islands subject to their rule, in order to secure the monopoly to Ambon.
During the Dutch period, Ambon city was the seat of the Dutch resident and military commander of the Moluccas. The town was protected by Fort Victoria, and a 1911 encyclopedia characterized it as "a clean little town with wide streets, well planted". The population was divided into two classes orang burger or citizens, and orang negri or villagers, the former being a class of native origin enjoying certain privileges conferred on their ancestors by the old Dutch East India Company. There were also, besides the Dutch, some Arabs, Chinese and a few Portuguese settlers.
Ambon city was the site of a major Dutch military base, which was captured from Allied forces by the Japanese in the Battle of Ambon (1942), during World War II. The battle was followed by the summary execution of more than 300 Allied POWs, in the Laha massacre.
Indonesia declared its independence in 1945. As a result of ethnic and religious tensions, as well as President Sukarno's making of Indonesia a centralised state, Ambon was the scene of a revolt against the Indonesian government, which resulted in the rebellion of Republic of the South Moluccas in 1950.