- Nicole McCallister (Jessica Bogart) - Harry's love interest and eventual girlfriend, Nicole is an attractive bookworm/archaeologist who came to the jungle to look for her father Kevin. She enlists in the help of an evil shaman named Pusca, who helped her find the temple where her father was last seen. Upon finding his lifeless body, Nicole becomes distraught and desperately searches for a loophole that will bring Kevin back. She is comforted by Harry, who sends her back to base camp, where she goes missing. Later, she returns to the Gate of El Dorado, and along with Harry and Micay, she places the artifacts in their slots and opens the gate before stealing them and revealing that she made a deal with the sorcerer Pusca: if she opened the gate, he was going to send her back six months in time to save her father. She is unaware that Pusca has no more use for her now and kills her before taking Micay and the artifacts through the time portal, and Harry goes in after them. After getting the magical powers from one of Micay's butterflies and defeating Pusca, Harry returns to the present, where her body lay. Using the same magic powers, he holds her, kisses her and brings her back to life, and the two are seen kissing at the very end of the game.
- Micay (Cree Summer-Francks) - the local native princess, who according to Harry is "Smokin' hot". In the game, he is required to "rescue" her. She loves butterflies and flirts with Harry on several occasions. She comes from the past and needed Harry to find all the artifacts so that she could save El Dorado and her people from the Spanish invaders. For his pains in rescuing her, he is given his shield.
- St. Clair (David Kaye) - Rival of Pitfall Harry. He has an unspecified history with Harry but there's is nothing but disdain between them. He is leading an Expedition to find El Dorado, working with Pusca. He is fought as a boss twice.
- Hubert Leech (Michael Beattie) - a young inventor with bad flying skills and a knack for getting stuck in bad places, for example, steep ledges and floating ice bergs. He is often seen using a flying contraption and gives harry the Heroic Handbook page for the "Heroic Dive", providing the dialogue.
Pusca (Michael Beattie) - An ancient Shaman who was around during the initial fall of El Doroado. He alligns himself with St. Claire but later kills him and takes command of his army. He is The last boss of the game.
- Quickclaw (Rino Romano) - a jaguar, with the power of speech. He doesn't really fit in, and follows Harry around just because he is lonely and doesn't fit in with the other jaguars. Quickclaw first met Harry in the jungle, but follows him all over the Amazon rainforest. He came from the past, like Micay, and used to steal food from the villagers. He was cursed with speech by Micay, so that he wouldn't fit in with mankind or his own kind. At the end of the game, Micay lifts the spell and Quickclaw is last seen walking off with his own kind.