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More than 220 kinds of birds have been seen, including:
- the elusive Mallee Fowl
- the rare Southern Scrub Robin Parrot
Animal life includes Brushtailed Possums and Sugar Gliders, kangaroos, bats and many kinds of reptiles. Stumpy-tailed Lizards and Bearded Dragons are often found basking in the sun.
More than 670 species of native plants have been recorded in the Little Desert, representing about one fifth of Victoria's indigenous flora.
The eastern block contains extensive heathlands, with banksia, tea-tree and sheoak, and many spring flowering species.
Woodlands of Yellow and Red Gum with Slender Cypress-pines, and swamps and clay flats of Bull-oak and melaleuca are of particular interest in the western block. Some twelve plant species are considered to be rare or significant.
The central block contains elements of the vegetation types of both the other blocks, with extensive areas of stringybark. Three plant species are considered rare or significant.
Scattered throughout sandy areas of the park are ridges of iron-rich sandstones on which Broombush can be found.