In July 1515, Willaert entered the Service of Cardinal Ippolito I d'Este of Ferrara. Ippolito was a traveler, and Willaert likely accompanied him to various places, including Hungary, where he likely resided from 1517 to 1519. When Ippolito died in 1520, Willaert entered the Service of Duke Alfonso of Ferrara. In 1522 Willaert had a post at the court chapel of Duke Alfonso; he remained there until 1525, at which time records show he was in the employ of Ippolito II d'Este in Milan.

Willaert was one of the most versatile composers of the Renaissance, writing music in almost every extant style and form. In force of personality, and with his central position as maestro di cappella at St. Mark's, he became the most influential musician in Europe between the death of Josquin and the time of Palestrina. Some of Willaert's motets and chanzoni franciose a quarto sopra doi (double canonic chansons) had been published as early as 1520 in Venice. Willaert owes much of his fame in sacred music to his motets.

From his appointment in 1527 until his death in 1562, he retained the post at St. Mark's. Composers came from all over Europe to study with him, and his standards were high both for singing and composition. During his previous employment with the dukes of Ferrara, he had acquired numerous contacts and influential friends elsewhere in Europe, including the Sforza family in Milan; doubtless this assisted in the spread of his reputation, and the consequent importation of Musicians from foreign countries into northern Italy. In Ferrarese court documents, Willaert is referred to as "Adriano Cantore". In addition to his output of sacred music as the Director of St. Mark's, he wrote numerous madrigals, a secular form; he is considered a Flemish madrigal Composer of the first rank.

With his contemporaries, Willaert developed the canzone (a form of polyphonic secular song) and ricercare, which were forerunners of modern instrumental forms. Willaert also arranged 22 four-part madrigals for voice and lute written by Verdelot. In an early 4-part vocal work, Quid non ebrietas? (In some sources called the Chromatic Duo) Willaert uses musica ficta around the circle of 5ths in one of the voices resulting in an augmented 7th in unison with the ending octave, an outstanding experiment with chromatic enharmonicism. Willaert was among the first to extensively use chromaticism in the madrigal. Looking forward, we are given an image of early word painting in his madrigal Mentre che'l cor. Willaert, who was fond of the older compositional techniques such as the canon, often placed the melody in the tenor of his compositions, treating it as a cantus firmus. Willaert, with the help of De Rore, standardized a five-voice setting in madrigal composition. Willaert also pioneered a style that continued until the end of the madrigal period of reflecting the emotional qualities of the text and the meanings of important words as sharply and clearly as possible.

Willaert was no less distinguished as a Teacher than as a Composer. Among his disciples were Cipriano de Rore, his successor at St. Mark's; Costanzo Porta; the Ferrarese Francesco Viola; Gioseffo Zarlino; and Andrea Gabrieli. Another Composer stylistically descended from Willaert was Lassus. These composers, except for Lassus, formed the core of what came to be known as the Venetian school, which was decisively influential on the stylistic change that marked the beginning of the Baroque era. Among Willaert's pupils in Venice, one of the most prominent was his fellow northerner Cipriano de Rore. The Venetian School flourished for the rest of the 16th century, and into the 17th, led by the Gabrielis and others. Willaert also probably influenced a young Palestrina. Willaert left a large number of compositions — 8 (or possibly 10) masses, over 50 hymns and psalms, over 150 motets, about 60 French chansons, over 70 Italian madrigals and 17 instrumental (ricercares).

He was born at Rumbeke near Roeselare. According to his student, the renowned 16th century music theorist Gioseffo Zarlino, Willaert went to Paris first to study law, but instead decided to study music. In Paris he met Jean Mouton, the principal Composer of the French royal chapel and stylistic compatriot of Josquin des Prez, and studied with him.