As a child Guillaume learned a good deal of science from his father, a clockmaker with a considerable scientific knowledge. Born in Fleurier, Switzerland, in 1878 he entered the Zurich Federal Institute of Technology, gaining his doctorate in 1882. In 1883 Guillaume became an assistant at the newly established International Bureau of Weights and Measures at Sèvres, near Paris. He was appointed director in 1915 and held this post until his retirement in 1936.

Guillaume's early work at the Bureau was concerned with thermometry; his treatise of 1889 on this subject became a standard text for metrologists. He was also involved in developing the international standards for the meter, kilogram, and liter. His research on thermal expansion of possible standards materials led him from 1890 to investigate various alloys. After a methodical study of nickel–steel alloys he devised an alloy that showed a very small expansion with temperature rise. Guillaume's new material (invar) found immediate practical applications, particularly in clocks, watches, and other precise instruments. He also produced a nickel–chromium–steel alloy, known as elinvar, with an elasticity that remains nearly constant over a wide range of temperatures. It became widely used, for example, for the hairsprings of watches.

In 1920 Guillaume received the Nobel Prize for physics for his researches into nickel–steel alloys.