At the time, disinfectant cleaners and topical antiseptic wound care were widely used but there were very few antimicrobial drugs to use safely inside living bodies.

This compound was first synthesized by Bayer chemists Josef Klarer and Fritz Mietzsch as part of a research program designed to find dyes that might act as antibacterial drugs in the body.

The molecule was tested and in the late autumn of 1932 was found effective against some important bacterial infections in mice by Gerhard Domagk, who subsequently received the 1939 Nobel Prize in Medicine.

The crucial test result (in a murine model of Streptococcus pyogenes systemic infection) that preliminarily established the antibacterial efficacy of Prontosil in mice dates from late December 1931.

To be convinced about it, it is enough to attentively examine the monthly reports of work of Mietzsch and Klarer during years 1935–1936 and especially the Log Book of Gerhard Domagk: the formula of sulphamide is consigned there – without comment – not before January 1936.

"[12] Dr. Alexander Ashley Weech (1895–1977), a pioneer pediatrician, while working at Columbia University's College of Physicians & Surgeons (in the affiliated New York Babies Hospital) treated the first patient in the United States with an antibiotic (sulfanilamide; prontosil) in 1935 which led to a new era of medicine across the Atlantic.

"[16] Dr. Perrin Long and Dr. Eleanor Bliss of Johns Hopkins University began their pioneering work later on prontosil and sulfanilamide which led to the large scale production of this new treatment saving the lives of millions with systemic bacterial infections.

Although quickly eclipsed by these newer "sulfa drugs" and, in the mid-1940s and through the 1950s by penicillin and other antibacterials that proved more effective against more types of bacteria, Prontosil remained on the market until the 1960s.