Tatum, who was born in Boulder, Colorado, studied chemistry at the University of Wisconsin, where his father was professor of pharmacology. He obtained the BA degree in 1931, then undertook research in microbiology for his master's degree, conferred the following year. His PhD was more biochemically oriented and after receiving his doctorate he worked as a research assistant in biochemistry for a year. He studied bacteriological chemistry at Utrecht University from 1936 to 1937 and on returning to America was appointed research associate at Stanford University.

His early experiments at Stanford concentrated on the nutritional requirements of the fruit fly, Drosophila melanogaster, but in 1940, in collaboration with George Beadle, he began working on the pink bread mold, Neurospora crassa. They irradiated the mold with x-rays to induce mutations and were then able to isolate a number of lines with different nutritional deficiencies. These lines needed special supplements to the basic growth medium to enable growth to continue as normal. When a mutant mold was crossed with the normal wild-type mold, the dietary deficiency was inherited in accordance with expected Mendelian ratios. Such studies established that genes act by regulating specific chemical processes. During World War II this work was of use in maximizing penicillin production, and it has also made possible the introduction of new methods for assaying vitamins and amino acids in foods and tissues.

In 1945 Tatum moved to Yale University where he extended his techniques to yeast and bacteria. Through studying nutritional mutations of the bacterium Escherichia coli, he and Joshua Lederberg were able to demonstrate, in 1946, that bacteria can reproduce sexually. Following this work, bacteria have become the primary source of information on the genetic control of biochemical processes in the cell.

Tatum returned to Stanford in 1948 and in 1957 joined the Rockefeller Institute for Medical Research. In 1958, together with Beadle and Lederberg, he received the Nobel Prize for physiology or medicine in recognition of the work that helped create the modern science of biochemical genetics.