Born in Fuenllana, Spain, in 1488, Thomas spent his childhood in Villanueva de los Infantes, from which he derived his surname. His parents, a miller, were known for their charitable acts, which deeply influenced Thomas from a young age. He pursued a superior education at the University of Alcalá, where he excelled, earning a degree in Theology and becoming a popular professor of philosophy, arts, and logic. Despite his academic success, he declined a prestigious professorship at the University of Salamanca, choosing instead to join the Augustinian friars in Salamanca in 1516, professing his vows in 1517 and being ordained a priest in 1518.

He was instrumental in sending the first Augustinian missionaries to the New World, specifically Mexico, in 1533, promoting both studies and missions within the Order. In 1544, after initially refusing the archbishopric of Granada, Thomas was nominated as Archbishop of Valencia. He reluctantly accepted the position in 1545.

He established programs where funds from the wealthy would support the poor, and he turned his own home into a soup kitchen and shelter, feeding hundreds daily.

Thomas died on September 8, 1555, in Valencia, at the age of 67, after suffering from angina. He was canonized by Pope Alexander VII on November 1, 1658. His liturgical feast day is celebrated on September 22. Saint Thomas of Villanova is the patron saint of Villanova University in Pennsylvania, among other educational institutions and parishes named in his honor.