The newly uncovered painting has been lying forgotten at the Nelson-Atkins Museum of Art in Kansas City, Missouri, since it was acquired in the 1930s.
Entitled The Temptation Of Saint Anthony, it shows the saint gathering water in a jug as he leans on a staff in what was probably part of a larger panel, possibly a triptych.
The surprise discovery comes as 's-Hertogenbosch, the hometown of the artist whose nightmarish visions on canvas earned him the moniker of "the devil's painter", marks the 500th anniversary of his death.
Initially it had been believed to be the work of one of the many students who flocked to Bosch's workshop in 's-Hertogenbosch.
But a team of international researchers who carried out a five-year research project using sophisticated infra-red technology determined that the painting was in fact by the master himself.
The canvas, which was likely painted between 1500 to 1510, "is a significant addition to the small body of existent work produced by Hieronymus Bosch," the researchers said in a statement.
"Although the image was heavily retouched and overpainted during a 20th century restoration, Bosch's hand is still clearly recognisable in the original brushwork," they said.
The painting was unveiled at the Noordbrabants Museum in Bosch's hometown which has succeeded in bringing together over 20 of his 25 or so surviving works for an unprecedented retrospective which opens on 13 February.
"The little monsters in the panel are typically 'Boschian'," the researchers said, highlighting a monster with a fox's head, and a toad clambering out of the water.
A floating sausage which can be seen in the painting is also included in other artworks by Bosch.
The newly discovered work will join 20 paintings and 18 other drawings among about 100 on display in the exhibit Hieronymus Bosch - Visions of Genius which runs until 8 May.
But Bosch's most famous work, a triptych called The Garden of Earthly Delights which journeys from Adam and Eve in the Garden of Eden to a terrifying vision of hell, will not be on display.
It hangs in the Museo Nacional del Prado in Madrid, and has never travelled abroad.