Mulerius was born Nicolaas des Mulier(s), son of Pierre des Muliers and Claudia Le Vettre. He grew up in Bruges, where he was taught by Cruquius, among others. From 1582 he studied medicine at the University of Leiden, where Lipsius, Vulcanius, Snellius and Heurnius were teachers. In 1589 he married Christina Six and set up practice for 13 years in Harlingen. In 1603 he became leading physician in Groningen, in 1608 he took the position as school master of the Leeuwardengymnasium. From 1614 he was professor in medicine and mathematics at the Groningen University. From 1619 – 1621 and 1626 – 1630, he was in charge of the library of the University of Groningen. His son, Petrus Mulerius (1599-1647), would become professor at Groningen in physics and botany from 1628.