Many ancient cultures, including those in Australia, China, Egypt, Greece and India, independently discovered the useful properties of fungi and plants in treating infections. These treatments often worked because many organisms, including many species of mould, naturally produce antibiotics. However, ancient practitioners could not precisely identify or isolate the active components in these organisms.
In England in 1640, the idea of using mould as a form of medical treatment was recorded by apothecaries such as the botanist John Parkinson, who documented the use of moulds to treat infections in his book on pharmacology. In 17th-century Poland, wet bread was mixed with spider webs (which often contained fungal spores) to treat wounds. The technique was mentioned by Henryk Sienkiewicz in his 1884 novel With Fire and Sword.
In 1871, Sir John Scott Burdon-Sanderson reported that culture fluid covered with mould would produce no bacterial growth. Joseph Lister, an English surgeon and the father of modern antisepsis, observed in November 1871 that urine samples contaminated with mould also did not permit the growth of bacteria. He also described the antibacterial action on human tissue of  Penicillium glaucum but did not publish his results. In 1875 John Tyndall demonstrated to the Royal Society the antibacterial action of the Penicillium fungus.
In 1876, German biologist Robert Koch discovered that a bacterium (Bacillus anthracis) was the causative pathogen of anthrax, which became the first demonstration that a specific bacterium caused a specific disease and the first direct evidence of germ theory of diseases. In 1877, French biologists Louis Pasteur and Jules Francois Joubert observed that cultures of anthrax bacilli, when contaminated with other bacteria, could be successfully inhibited. Reporting in the Comptes Rendus de lAcadémie des Sciences', they concluded:
The phenomenon was described by Pasteur and Koch as antibacterial activity and was named antibiosis by French biologist Jean Paul Vuillemin in 1877. (The term antibiosis, meaning against life, was adopted as antibiotic by American biologist and later Nobel laureate Selman Waksman in 1947.) However, Paul de Kruifs 1926 Microbe Hunters' notes that Pasteur believed that this was contamination by other bacteria rather than by mould. In 1887, Swiss physician Carl Garré developed a test method using glass plate to see bacterial inhibition and found similar results. Using his gelatin-based culture plate, he grew two different species of bacteria and found that their growths were inhibited differently, as he reported:
In 1895, Vincenzo Tiberio, an Italian physician at the University of Naples, published research on moulds initially found in a water well in Arzano; from his observations, he concluded that these moulds contained soluble substances having antibacterial action. Two years later, Ernest Duchesne at École du Service de Santé Militaire in Lyon independently discovered the healing properties of a P. glaucum mould, even curing infected guinea pigs of typhoid. He published his results in a dissertation in 1897. Duchesne was using a discovery made earlier by Arab stable boys, who used moulds to cure sores on horses. He did not claim that the mould contained any antibacterial substance, only that the mould somehow protected the animals. Penicillin does not cure typhoid and so it remains unknown which substance might have been responsible. A Pasteur Institute scientist, Costa Rican Clodomiro Picado Twight, similarly recorded the antibiotic effect of Penicillium in 1923. In these early stages of penicillin research, most species of Penicillium were non-specifically referred to as P. glaucum, so that it is impossible to know the exact species and that it was really penicillin that prevented bacterial growth.
Andre Gratia and Sara Dath at the Free University of Brussels studied the effects of mould samples on bacteria. In 1924, they found that dead Staphylococcus aureus cultures were contaminated by a mould, a streptomycete. Upon further experimentation, they showed that the mould extract could kill not only S. aureus, but also Pseudomonas aeruginosa, Mycobacterium tuberculosis and Escherichia coli. Gratia called the antibacterial agent "mycolysate". The next year they found another killer mould that could inhibit B. anthracis. Reporting in , they identified the mould as P. glaucum. But these findings received little attention as the antibacterial agent and its medical value were not fully understood, and Gratia's samples were lost.