Argotti's history as a Botanic Garden originates as a physic garden that included herbs and medicinal plants. That was established around 1674 by the Knights of St John at Fort St Elmo near the Sacra Infermeria.

When the British arrived in Malta, the medicinal plants and other botanical specimens were moved from St Elmo to Il-Mall in Floriana under the guidance of the Carmelite monk and Professor of Botany, Carolus
Giacinto who was appointed Chair of Natural History by the University of Malta in 1805. After Giacinto died in 1855, the botanic gardens were relocated to Argotti Gardens by Professor Stefano Zerafa who was the Professor of Natural History at that time.

Argotti Botanic Gardens & Resource Centre initially started as two separate private gardens that were built in the early 18th century. One was owned by the Knight Don Emmanuel Pinto de Fonseca and the other by Bailiff Ignatius de Argote y Gusman. In 1741, after Pinto's election to Grandmaster, De Argote purchased Pinto's part of the garden and annexed it. Argote built a nymphaeum with a statue of Diana in an ornamental grotto covered by pieces of red coral, calcite crystals, seashells and coloured pebbles. During this period, the formal botanic garden was not yet established.

Zerafa's contribution to the gardens' botanical assets was very significant. Apart from writing the first account on Maltese flora, "Thesaurus of the Flora of the Maltese Islands", he is mostly remembered for the discovery and naming of our national plant, the endemic Cheirolophus crassifolius - the Maltese rock centaury. In 1890 that the collection at Argotti Botanical Gardens was officially recognised as a botanical garden, making it the third oldest in the Commonwealth, after that of Oxford (1632) and Edinburgh (1670). The gardens enhanced their collections through contributions by leading Maltese botanists prominent amongst which were Count Alfred Caruana Gatto and Professor John Borg. This resulted in notable collections of indigenous and Mediterranean flora, cacti and succulents.

The gardens were passed over to the Department of Agriculture in 1973 after 150 years of university tenure. The inner part was returned to the University of Malta in 1996, and, since then, the University has been involved in conservation and propagation of rare species, including endemics and also serves as a means of education for researchers and the general public.

In August 2023, Argotti Botanic Gardens & Resource Centre attained BGCI (Botanic Gardens Conservation International) that distinguishes botanic gardens and recognizes achievements in plant conservation and sustainability.