After qualifying as a doctor, Fleming joined the Inoculation Department at St. Mary's Hospital, under the direction of Sir Almroth Wright, a pioneer in vaccine therapy. This marked the beginning of his long and distinguished research career.

During World War I, Fleming served as a captain in the Royal Army Medical Corps. He worked in battlefield hospitals in France, where he witnessed the devastating effects of wound infections and became disillusioned with the antiseptic methods then in use, as they often did more harm than good.

Fleming returned to St. Mary's after the war and continued his research on antiseptics and wound infections. His wartime experiences had strengthened his resolve to find more effective ways to combat bacterial infections.

Fleming discovered lysozyme, an enzyme found in body fluids that has antibacterial properties. While lysozyme proved to have limited antibacterial effectiveness, this discovery demonstrated Fleming's keen observational skills and his interest in naturally occurring antibacterial substances.

In September 1928, Fleming made his most famous discovery. Upon returning to his laboratory after a vacation, he noticed that a petri dish containing "Staphylococcus" bacteria had been contaminated by a mold. Around the mold, the bacteria had been killed. He identified the mold as "Penicillium notatum" and named the antibacterial substance it produced penicillin.