In 1916 London, Dr. Lily Houghton's Tears of the Moon research is presented by her brother MacGregor to the Royal Society. The Houghtons, hoping to revolutionize both medicine and the British War Effort, request access to a recently acquired arrowhead artifact, but the request is denied as the Tree is considered a myth and female scientists are disfavored. However, believing the arrowhead and her old Amazon map are the key to finding the Tree, Lily steals it, narrowly evading Prince Joachim, who is equally intent on finding the Tree for Germany.

Arriving in Brasil, Lily and MacGregor search for a guide to navigate the Amazon River. They hire skipper Frank Wolff who offers jungle cruises embellished with faked theatrical dangers and corny puns. He initially declines, citing the dangers of the river and jungle, but reconsiders upon seeing the arrowhead. Frank steals back his repossessed boat engine from the harbormaster Nilo and the trio departs after escaping from Joachim's U-boat.

In Frank's cabin, Lily finds photos and sketches of modern inventions as well as research on the Tears of the Moon. She accuses him of seeking the Tree, but he insists he gave up long ago. They are captured by the Puka Michuna tribe, disguised as cannibals, but quickly release as they were hired by Frank. Angered, Lily begins to doubt Frank. The tribal chief Trader Sam translates the symbols on the arrowhead, revealing the Tree's location and that it only blooms under a blood moon.

Meanwhile, Joachim has located the conquistadors petrified inside a cave. He makes Aguirre agree to find the arrowhead for him in exchange for flowers. Joachim diverts the river to free them as Aguirre and his conquistadors are reanimated while fused with rainforest elements. The conquistadors track down and attack the tribe where Frank is stabbed through the heart by Aguirre. Lily flees with the artifact, but vines pull the conquistadors away when they unknowingly lose sight of the river while pursuing her.