As a Calvinist, he was forced to leave Munich in 1591 when a rule was imposed that all members of the court had to proclaim their adherence to the Catholic faith. Hoefnagel refused to do so. He then went to work for Emperor Rudolf II, first residing in the city of Frankfurt am Main, where he moved in a circle of Flemish humanists, merchants, artists and publishers. In particular his friendship with the Flemish botanist Carolus Clusius may have played an important role in his later botanical illustrations. In 1594, he was forced to leave Frankfurt because of the repression of the Calvinist faith. He worked in his final years in Vienna but made regular visits to Prague. His brother Daniel lived in Vienna, where he ran a business under court protection. At this time Joris Hoefnagel promoted his son Jacob at court. He regularly collaborated with his son on artistic projects. According to Karel van Mander he died in Vienna in 1600 but this is not certain as there continue to be references to him in documents referring to his brother Daniel and his son Jacob after that date.