Guillaume was born in Edmonton, Middlesex, the son of Alfred Guillaume. He took up Arabic after studying Theology and Oriental Languages at the Wadham College, Oxford. In the First World War, he served in France and then in the Arab Bureau in Cairo. Guillaume was a Christian and later ordained.
He became Professor of Arabic and the Head of the Department of the Near and Middle East in the School of Oriental and African Studies (SOAS), in the University of London. He was later Visiting Professor of Arabic at Princeton University, New Jersey. He was a professor of Hebrew at Durham University from 1920-30.
In the winter 1944-45, during the Second World War the British Council invited him to accept a visiting professorship at the American University of Beirut where he greatly enlarged his circle of Muslim friends. The Arab Academy of Damascus (1949) and the Royal Academy of Baghdad (1950) honoured him by electing him to their number, and the University of Istanbul chose him as their first foreign lecturer on Christian and Islamic theology. In the Autumn of 1945, Guillaume succeeded his friend S. H. Hooke on the Samuel Davidson chair at the University of London, changing to the chair in Arabic in 1947 (at SOAS), and was also a professor of Hebrew from 1947–55. In 1955, Guillaume served as president to the Society for Old Testament Study.
He was best known as the author of Islam, published by Penguin Books, and as co-author, with Sir Thomas Arnold, of The Legacy of Islam, in the Legacy series, which has been translated into several languages. He also translated Ibn Ishaq's "Sirah Rasul Allah", published as The Life of Muhammad. A translation of Ishaq's "Sirat Rasul Allah".
- The Life of Muhammad. A translation of Ibn Ishaq's Sirat Rasul Allah ISBN 0-19-636034-X Oxford 1955
- The traditions of Islam : an introduction to the study of the Hadith literature ISBN 978-0-8369-9260-1 Oxford: Clarendon Press, 1924
- (with Thomas Arnold) The Legacy of Islam ISBN 978-81-7151-239-3 Oxford, Clarendon Press, 1931
- Kitāb Nihājat al-iqdām fī ʿilm al-kalām / Abu-ʾl-Fatḥ Muḥammad Ibn-ʿAbd-al-Karīm aš- Šahrastānī Oxford University Press, 1934
- Prophecy and Divination Among the Hebrews and Other Semites Bampton Lectures London: Hodder & Stoughton, 1938
- Hebrew and Arabic lexicography Leiden: Brill, 1965
- Islam ISBN 978-0-14-013555-8 Hammondsworth, Penguin 1954
- "Professor Alfred Guillaume: Arabic and Islamic Scholar". The Times. The Times Digital Archive. 3 December 1965. p. 16.
- Smith, Sidney (1966-01-01). "Obituary: Alfred Guillaume". Bulletin of the School of Oriental and African Studies, University of London. 29 (2): 478–481. doi:10.1017/s0041977x00060080. JSTOR 611301.