By around 1600, the Republic had become a great power. Back in 1596, France and England had recognised the Republic by forming a triple alliance with it. As a result, the war with Spain became part of a wider anti-Habsburg campaign headed by France. In 1600, Prince Maurice, who was both Stadholder and commander of the Republic's land and sea forces, was instructed by the States General and the great merchants to occupy the towns along the Flemish coast and extirpate the pirates based at Dunkirk, who posed a constant threat to the Republic's growing merchant fleet. Maurice undertook the expedition very much against his better judgement. His forces clashed with those of Archduke Albert, governor-general of the southern Netherlands and son-in-law of Philip II of Spain, in the dunes at Nieuwpoort. Maurice managed to win the battle but was unable to fulfil his mission of seizing the Flemish towns and Dunkirk. At sea, too, the war continued, with Jacob van Heemskerck overcoming a Spanish fleet near Gibraltar in 1607. This was the first major naval victory by the Republic and was of great strategic value. Van Heemskerck himself perished during the fighting.

In 1608 Spain and the Republic held peace talks in The Hague, with England and France present as mediators. This culminated in the signing of the Twelve Years' Truce in 1609. The issue led to a confrontation between the two most powerful men in the Republic: the Stadholder, Prince Maurice, would have preferred hostilities to continue, while the Grand Pensionary, Johan van Oldenbarnevelt, championed peace. It was not the only issue dividing them. Within the Reformed Church, a bitter conflict had broken out between two groups calling themselves Remonstrants and Counter-Remonstrants. The prince took the side of the Counter-Remonstrants, who advocated doctrinal orthodoxy, while Oldenbarnevelt supported the more moderate Remonstrants. Feelings ran high and civil war seemed imminent. The Counter-Remonstrants emerged victorious from the Synod of Dordrecht in 1618. Subsequently, a specially constituted tribunal found Oldenbarnevelt guilty of high treason and he was executed in The Hague on 13 May 1619. The religious controversy, and particularly the attitude adopted by Prince Maurice, inflicted lasting damage on the relationship between the States General and the House of Orange.

The Twelve Years' Truce expired in 1621. An initial desire to replace the Truce by a definitive peace treaty foundered in the face of unacceptable demands on the Spanish side. On 23 April 1625, Prince Maurice died. He was succeeded by his half-brother Frederick Henry, Count of Nassau and Prince of Orange, William of Orange's youngest son born to his fourth wife Louise de Coligny. Frederick Henry breathed new life into the conflict with Spain, taking many towns from the Spanish and thereby earning the name 'stedendwinger' (conqueror of towns). He owed his military training to Prince Maurice and Simon Stevin, a mathematical engineer employed by the Stadholder.

In 1639 a second Spanish Armada was dispatched to the Low Countries with 20,000 men on board, in a further attempt to subdue the rebel States. Admiral Maarten Tromp sailed against it with a much smaller fleet and destroyed the Armada in the Battle of the Downs.