Following the 1547–49 war with the Toungoo, Ayutthaya king Maha Chakkraphat built up his capital city's defenses in preparation for a later war with the Burmese. The 1547–49 war ended in a Siamese defensive victory and preserved Siamese independence. However, Bayinnaung's territorial ambitions prompted Chakkraphat to prepare for another invasion. These preparations included a census that prepared all able men to go to war. Arms and livestock were taken by the government in preparation for a large-scale war effort, and seven white elephants were captured by Chakkraphat for good luck. News of the Ayutthayan king's preparation spread quickly, eventually reaching the Burmese.

Bayinnaung succeeded in taking the city of Chiang Mai in the nearby Lan Na kingdom in 1556. Subsequent efforts left most of northern Siam under Burmese control. This left Chakkraphat's kingdom in a precarious position, faced with enemy territory to the north and the west. Bayinnaung subsequently demanded two of King Chakkraphat's white elephants as tribute to the rising Toungoo Dynasty. Chakkraphat refused, leading to Burma's second invasion of the Ayutthaya Kingdom.

The Bayinnaung armies marched down to Ayutthaya. There, they were kept at bay for weeks by the Siamese fort, aided by three Portuguese warships and artillery batteries at the harbor. The invaders finally captured the Portuguese ships and batteries on 7 February 1564, after which the fort promptly fell.^[43] With a now 60,000 strong force combined with the Phitsanulok army, Bayinnaung reached Ayutthaya's city walls, heavily bombarding the city. Although superior in strength, the Burmese were not able to capture Ayutthaya, but demanded that the Siamese king come out of the city under a flag of truce for peace negotiations. Seeing that his citizens could not take the siege much longer, Chakkraphat negotiated peace, but at a high price.

In exchange for the retreat of the Burmese army, Bayinnaung took Prince Ramesuan (Chakkraphat's son), Phraya Chakri, and Phraya Sunthorn Songkhram back with him to Burma as a hostage, and four Siamese white elephants. Mahathamraja, although a betrayer, was to be left as ruler of Phitsanulok and viceroy of Siam. The Ayutthaya Kingdom became a vassal of the Toungoo Dynasty, required to give thirty elephants and three hundred catties of silver to the Burmese yearly.