He was born in Pont-à-Mousson, Lorraine, France, where his father, William Barclay, held the chair of civil law. His mother was a Frenchwoman. His early education was obtained at the Jesuit College. While there, at the age of nineteen, he wrote a commentary on the Thebais of Statius. In 1603 he crossed with his father to London. Barclay had persistently maintained his Scottish nationality in his French surroundings, and probably found in James VI and I's accession an opportunity which he would not let slip.

He did not remain long in England, where he is supposed to have published the first part of his Euphormionis Satyricon against the Jesuits, for in 1605 when a second edition of that book appeared in Paris, he was there, having already spent some time in Angers, and being now the husband of a French girl, Louise Debonaire. He returned to London with his wife in 1606, and there published his Sylvae, a collection of Latin poems. In the following year the second part of the Satyricon appeared in Paris. James VI is said to have been attracted by his scholarship, but particulars of this, or of his life in London generally, are not available. In 1616 he went to Rome, for some reason unexplained, and there resided till his death on the 15th of August 1621.

In 1609 he edited the De Potestate Papae, an anti-papal treatise by his father, who had died in the preceding year, and in 1611 he issued an Apologia or "third part" of the Satyricon, in answer to the attacks of the Jesuits. A so-called "fourth part," with the title of Icon Animorum, describing the character and manners of the European nations, appeared in 1614.

He appears to have been on better terms with the Church and notably with Bellarmine; for in 1617 he issued, from a press at Cologne, a Paraeneis ad Sectarios, an attack on the position of Protestantism. The literary effort of his closing years was his best-known work the Argenis, a political romance, resembling in certain respects the Arcadia of Sidney, and the Utopia of More, completed about a fortnight before his death, which has been said to have been hastened by poison.