The document, acquired by Les Enluminures Gallery in New York, shows a carefully-drawn sketch of a kangaroo (know as a "canguru" in Portuguese) in its text and is dated between 1580 and 1620.
It has led researchers to believe images of the marsupial were already being circulated by the time the Dutch ship Duyfken - long thought to have been the first European vessel to visit Australia - landed in 1606.
The European discovery of Australia has been credited to the Dutch voyage headed by Willem Janszoon in 1606, but historians have suggested the country may already have been explored by other Western Europeans.
"It is not surprising at all that an image of a kangaroo would have turned up in Portugal at some point in the latter part of the 16th century, it could be that someone in the Portuguese expedition had this manuscript in their possession," Trickett said.
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Les Enluminures Gallery acquired the pocket-sized manuscript from a rare book dealer in Portugal.
"A kangaroo or a wallaby in a manuscript dated this early is proof that the artist of this manuscript had either been in Australia or even more interestingly, that traveller's reports and drawings of the interesting animals found in this new world were already available in Portugal," Les Enluminures researcher Laura Light said.
National Library of Australia curator of maps Martin Woods said while the image looked like a kangaroo or a wallaby, it alone was not proof enough to alter Australia's history books.
"The likeness of the animal to a kangaroo or wallaby is clear enough, but then it could be another animal in south-east Asia, like any number of deer species some of which stand on their hind legs to feed off high branches," Woods said.
Also nestled in letters of the text are two male figures adorned in tribal dress, baring naked torsos and crowns of leaves, which Light said could be depictions of Australian or south-east Asian natives.