The first part of the garden was bought in 1550 by Eleonora di Toledo, the wife of Cosimo I de' Medici, from the Pitti family. The landscaping was carried out by Niccolò Pericoli detto Tribolo; after his death in 1555, works were directed by Davide Fortini followed by Giorgio Vasari.
Ammannati also contributed to the project between 1560 and 1583, designing the courtyard that still bears his name.
The large semi-elliptical area known as the Amphitheatre, which was later to create a harmonious whole with the two wings of Ammanati's courtyard, was dug out of a huge stone quarry at the foot of the Belvedere hill. This architectural feature serves to visually unify the Pitti Palace and Boboli Gardens.

After 1574 Francis I called upon the help of the architect Bernardo Buontalenti, who designed the Grotta Grande. The grotto was created by transforming a fish pond built between 1556 and 1560 following the design of Giorgio Vasari.
The niches at the sides of the entrance to the grotto hold the statues of Bacchus and Ceres by Baccio Bandinelli (1552-1556). Until 1924, when they were replaced with cement casts, Michelangelo's Prisoners stood in the corners.
During the early 17th century, the period of the grand duchy of Cosimo I (1609-1621), the garden was extended beyond the wall built during the war against Siena under the supervision of Gherardo Mechini and Giulio Parigi.
The ideal axis of the garden is the cypress avenue leading to the impressive Bacino dell'Isola (Island Basin), built between 1612 and 1620. Giulio Parigi was also responsible for the Vasca dell'Isola (Island Pond), one of the most suggestive spaces in the garden originally intended for the cultivation of citrus fruits and flowers.
Originally at the centre of the pond was a Venus fountain, replaced by Giambologna's Oceanus in 1636 upon the orders of Grand Duke Ferdinando II.
During that same year, the statue of Plenty, begun by Giambologna and completed by Pietro Tacca was placed in its current position.

In the 18th century, the Medici dynasty disappeared and the Grand Duchy passed to the Hapsburg-Lorraine family. After an initial period of abandonment, widescale restoration work took place under Pietro Leopoldo of Lorraine (1765-1790) involving the sculptures, architecture, waterworks and plants.
New buildings were also built in the garden including the Kaffeehaus (1775) and the Limonaia (a building for raising citrus fruits constructed from 1777-1778), designed by Zanobi del Rosso, and the Palazzina della Meridiana, begun in 1776 by Niccolò Gaspero Paoletti.
A new phase of decadence took place during the period of Napoleonic rule (1799-1814) and again after the failed attempt of the Grand Duchess Elisa Baciocchi to transform Boboli into an English-style garden.
The restoration under the Lorraine family restored the original formal appearance of the Boboli Gardens. In 1834, under Leopoldo II, the labyrinths were destroyed to make way for a wide avenue suitable for carriages, following the design of Pasquale Poccianti.
During the 19th century, the garden provided the backdrop for spectacular open-air entertainment.