The Museum had specimen from planets from the Mutter's Spiral and the Andromeda Galaxy, including Earth, Mars, Venus, Mondas, Raxacoricofallapatorius, Refusis II, Varos and Tara. These specimen were kept in stasis in clear boxes. Teams were devoted to collecting from these planets, generally chosen to resemble the natives. They had pendants which allowed them to teleport to the planet and communicate with the other members of the team. Eve had a machine which could sense when a species was going extinct and where, allowing her to send in teams to collect the last specimen. The machine made logical prediction and couldn't predict the future, so she was unable to react quickly enough if the whole planet was destroyed. Time travelling species, like the Daleks and Time Lords, were hard to track and eventually Eve stopped trying to follow them.
The museum occupied a whole planet and the hemispheres were open to the public in shifts. Because of the museum's great size, a comprehensive system of teleports was used to speed up travel across the planet. There was also a gift shop and cafeteria with vending machines instead of cafeteria workers. (PROSE: The Last Dodo)
Around 500 million BC, Hr'oln survived the destruction of his planet and found a new planet to live on. He created Eve to interact with the humanoid inhabitants. When these inhabitants were wiped out by a plague, he programmed Eve to find a way to save other species. Eve decided the best way to do that would be to capture the last of every species and kept them in stasis, as she did with Hr'oln. She created the Museum of the Last Ones to store these individuals.
Around the 2060s, Eve and Frank started cloning specimens to sell to pay for the museum. Eve was also creating clones of dodos and sabre-toothed tigers, which she would use to destroy entire planets. The sabre-toothed tigers would cause havoc, while the dodos would plant bombs. Using this method, Eve planned to destroy all life in the universe except for those stored in the museum.
In 2062, Frank wanted to make more money, so he stole specimens and sold them on Earth. When Martha Jones and the Tenth Doctor came to the Museum to complete the I-Spyder Book of Earth Creatures, they were blamed. The Doctor and Martha were able to find out who really stole them, but when Eve found out the Doctor was the last Time Lord, she captured him for the museum. When Martha tried to free him with the sonic screwdriver, she accidentally sent all the other Earth creatures back to Earth. Eve took advantage of this chaos and sent in sabre-toothed tigers and dodos with bombs to destroy Earth. The Doctor later confronted Eve and deactivated her by tampering with the gun she was using, causing it to backfire. He then deactivated all the bombs on Earth and modified the teleport, sending all the animals at the Museum back in time to before they went extinct. (PROSE: The Last Dodo)