Four trees in the Gardens are included on the National Trust (Victoria) list of significant trees. These are:

African Sweet Thorn (Acacia karroo). This is listed as being of State significance (T11374). It is one of several African trees in the Gardens, but is now dying. It is estimated to be about 80 years old. However, it is formally declared as a noxious weed and has been removed from almost every other Botanic Gardens in the country. This specimen does not appear to have produced seeds.

Prickly Bottlebrush (Callistemon brachyandrus). This species, with its showy flowers, is native to NSW, SA, and Victoria. The specimen is of State significance (T11376). However, it has been damaged in recent years and is in quite poor condition.

Kei Apple (Dovyalis caffra). This is listed as being of State significance (T11375). This southern African tree is the best specimen of the species in the State: the only others are one in Werribee Park and seven in the Royal Botanic Gardens, Melbourne. The fruits are made into jam by some of the Friends.

Soledad Pine (Pinus torreyana). This is listed as being of State significance (T11377). It naturally occurs at only two locations on Santa Rosa Island and on the San Diego Coast, California.

Four other trees are also listed in the Heritage Council's registration of the Gardens: a Long-Leaved Indian Pine (Pinus roxburghii) from from the Himalayan foothills; a Pencil Juniper (Juniperus virginiana) from North America; a Cabbage Palm ( Livistonia australis ) from Eastern Australia; and a Brazilian Pepper Tree (Schinus terebinthifolius) from Southern Brazil, Paraguay and Argentina.

Other notable trees include a very large Stone Pine (Pinus pinea) from Portugal and Spain which produces edible pine nuts, an Aleppo Pine (Pinus halepensis), an Italian Cypress (Cupressus sempervirens), a Moreton Bay Fig (Ficus macrophylla), a Red Ironbark (Eucalyptus sideroxylon) and remnant River Red Gums (Eucalyptus camaldulensis), Yellow Box (Eucalyptus melliodora) and Grey Box ( Eucalyptus macrocarpa). Sugar Gum (Eucalyptus cladocalyx), Dutch Elms (Ulmus x hollandica), and one remaining Blue Gum (Eucalyptus globulus subsp. globulus) first planted in 1869 are also represented in the gardens.

A list of all the trees and shrubs in the gardens can be obtained by clicking here.