Born the son of a basket maker at Delft in the Netherlands, Leeuwenhoek received little formal education and was apprenticed to a linendraper at the age of 16. In about 1654 he set up in business in Delft as a draper. He also served from 1660 as chamberlain of the town's law courts.

In 1673 Henry Oldenburg, secretary of the Royal Society, received a letter from a Dutch correspondent informing him that “a certain most ingenious person here Leeuwenhoek has devised microscopes which far surpass those manufactured by others.” A letter from Leeuwenhoek describing his observations of bees, mold, and lice was enclosed. It was published in the Philosophical Transactions of the Royal Society in 1673. It was the first of 165 letters reporting Leeuwenhoek's observations which would appear in the Transactions between 1672 and his death in 1723. Leeuwenhoek wrote no books and, lacking Latin, reported his work in Dutch, which was then translated into English or Latin for publication.

Among the highlights of the Letters are his 1674 observations of his ‘little animalcules’ (protozoa) discovered in rainwater that had stood for a few days. They were, he estimated, some 10,000 times smaller than water fleas. He also gave some idea of the profusion of nature by calculating that “there were upwards of 1 million living creatures in one drop of pepper-water.” He was sufficiently detached to examine with his microscope his own faeces and note that “when of ordinary thickness” no animalcules were observed but whenever “the stuff was a bit looser than ordinary I have seen animalcules therein.”

Leeuwenhoek also announced in 1679 his discovery of human spermatazoa. In 1677 a Mr Ham brought him “the spontaneously discharged semen of a man who had lain with an unclean woman and was suffering from gonorrhea.” He observed little animals within, “animalcula in semine masculino” (animalcules in human semen), and noted they had tails and lived for a few hours only. He went on to examine the sperm of birds, frogs, insects, cattle, and several other species. A further important biological observation was Leeuwenhoek's 1684 description of red blood cells, which he estimated to be 25,000 times smaller than a fine sand grain.

Leeuwenhoek's instruments were all simple microscopes with a single small lens clamped between two metal plates. The object was placed on a fine pin and its distance from the plates adjusted by turning a screw. On his death he left 247 completed microscopes and 172 mounted lenses. They were auctioned and dispersed in 1747. A further 26 mounted in silver were bequeathed to the Royal Society but disappeared without trace in the mid-nineteenth century. Nine of Leeuwenhoek's original microscopes have survived, with a highest magnification of 266 and resolution of 2 micrometers.

Towards the end of his life Leeuwenhoek became something of a European celebrity. Monarchs such as Peter the Great and Queen Mary traveled to Delft to be shown the ‘little animalcules’ by Leeuwenhoek himself.