He studied at the University of Glasgow and at Berlin and Leipzig.
In Germany MacCallum concentrated on medieval literature, he published several articles in the Cornhill Magazine in 1879-1880. In 1884 he published Studies in Low German and High German literature. MacCallum became Professor of Literature at the University of Wales, Aberystwyth in 1879, but moved to Sydney in 1887 to take up the post of Foundation Professor of Modern Language and Literature at Sydney University, MacCallum was chosen over 44 other candidates.
In 1897 MacCallum became president of the Sydney University Union.
In 1898 he was made Dean of the Faculty of Arts. In April 1928 MacCallum was elected deputy chancellor and became Chancellor of the university in 1934.
The Mungo MacCallum Building at the University of Sydney was named in his honour. Sir Mungo wrote a number of works of literary criticism on English and German literature, and is most notable for his work on Shakespeare.
In 1894, MacCallum published a book Tennyson"s Idylls of the King and Arthurian Story from the 16th century in which he traced the Arthurian story from its "Brythonic" origins through Thomas Malory and up to its final phase in Lord Tennyson.
In his 1967 foreword to Shakespeare"s Roman Plays and Their Background, Terence Spencer of the Shakespeare Institute judged MacCallum"s "indispensable" 1910 book as unusual in having "outlasted changes of fashion in criticism.".