The Museum was officially instituted in 1842, when Filippo Parlatore compiled in the Museo di Fisica e Storia Naturale the Herbarium Centrale Italicum, the first national botanical collection of herb samples. In 1854 the museum acquired the great herbarium of the British traveller and botanist, Philip Barker Webb, containing many samples from the 18th and 19th centuries coming from Africa, South America and Oceania. In the late 19th century the Museum was moved to its new location in Via La Pira, where it was enriched with new acquisitions (the Beccari Herbarium from Malaysia and the Libyan Herbarium). The Botanical Museum is today the most important of its kind in Italy, both for the extent of its collections (around 4 million specimens) and for their scientific value.

The collection of herbariums includes: the Herbarium Centrale Italicum (phanerogamic and critogamic section 3,600,000 specimens in all), Webb Herbarium (300,000 items), Beccari Herbarium from Malaysia (16,000 specimens), Micheli-Targioni Tozzetti Herbarium (19,000 specimens), historical Herbariums (Cesalpino, Merini and others: 2,000 specimens).

The collection of botanical wax models, most of them fabricated between the end of he 18th century and the middle of the 19th by famous wax-modellers such as Francesco Calenzuoli, Luigi Calamai and Egisto Tortori, is composed of life-size models of plants and fruits (466 objects, a few of them made of plaster or papier-maché) and specimens prepared on wood illustrating the anatomy, physiology and pathology of plants (38 plates). The Museum also includes seventeenth-eighteenth century paintings of botanical subjects by the Medicean painter Bartolomeo Bimbi and by Filippo Napolitano, as well as eighteenth-century copies of the works of Bimbi (about 50 pictures).

The botanical collection includes the palynology section (400 specimens in test tubes and microscopic preparations), wood samples (7,000 items), the carpophore section (16,000 specimens, dried or conserved in alcohol), and the seed section (240 specimens in test tubes), as well as plant fossils (about 10,000 specimens).