Goldberger, the son of Jewish immigrants, was brought to America at the age of six. He was educated at the College of the City of New York and at Bellevie Hospital Medical School. After a brief period in private practice Goldberger joined the US Public Health Service in 1899, remaining there for the rest of his life.

Goldberger worked as a field officer for many years, making contributions to the understanding and control of such diseases as yellow fever, typhus, and dengue. He is, however, mainly remembered for his authoritative investigation of the nature, causation, and treatment of pellagra. This disease, which became widely known in America after the Civil War, is typified by chronic diarrhea, roughening of the skin, a sore tongue, and involvement of the nervous system. Death from secondary infection or general emaciation was not uncommon.

When Goldberger began his work in 1913 it was thought that the disease was caused by an unknown toxin produced by bacterial fermentation during storage of grain. But stimulated by the work of Frederick Gowland Hopkins and Casimir Funk, Goldberger directed his attention to deficiency diseases. He began a classic investigation into the connection between pellagra and diet in various asylums and orphanages of the southern states. He was immediately struck by the fact that the staff of such institutions – with a diet containing milk, eggs, cheese, and meat – remained free of the disease while the inmates, subsisting virtually on cereals alone, frequently suffered from epidemics of pellagra.

It was a relatively simple matter to show that the disease could be eliminated by supplementing the inmates' diet with milk. He was further able to trade the offer of a pardon with 11 inmates of a Mississippi prison for their adoption of a diet of corn, rice, sugar, pork fat, potatoes, and turnips. Within a few months 7 of the 11 were showing early symptoms of pellagra. Attempts to transmit the disease by contact with the clothes, excreta, and vomit of the patients ended in failure. Whatever such a factor might be, he was able to show by 1920 that sufficient of it was contained in a daily dose of 15–30 grams of yeast, and by this means alone Goldberger was able to prevent the 10,000 deaths a year attributable to pellagra in the USA.

The active ingredient involved was shown in 1937 by Conrad Elvehjem (1901–1962) to be nicotinic acid (niacin), part of the vitamin B complex.