Perfect Dark is set in 2023 against the backdrop of an interstellar war between two alien races: the Maians, who resemble the archetypal Grey alien, and the Skedar, reptile-like creatures who use a cloaking device to appear human. On Earth, there is an ongoing rivalry between two companies: The Carrington Institute, a research centre founded by Daniel Carrington that secretly operates an espionage group in league with the Maians; and dataDyne, a defence contractor corporation headed by Cassandra de Vries. In exchange for creating an AI with code-breaking abilities to access an ancient alien spacecraft at the bottom of the Pacific Ocean, the Skedar agree to supply dataDyne with enough alien technology to become the biggest corporation on Earth.
The player is cast as Joanna Dark, an agent of the Carrington Institute whose excellent scores in training have earned her the codename "Perfect Dark"On her first mission, she is sent to extract a defector known as Dr. Caroll from a dataDyne laboratory. Dr. Caroll is revealed to be the AI created by dataDyne, and is worried about the mission for which it had been designed. After the extraction, Carrington is held captive at his private villa by dataDyne soldiers. When Joanna rescues him, she is informed that Dr. Caroll has been taken to a dataDyne front in Chicago. There, Joanna learns that Cassandra, NSA director Trent Easton, and a mysterious man known as Mr. Blonde plan to kidnap the President of the United States to get access to a deep sea research vessel called the Pelagic II. Although the President is in danger, Carrington alerts Joanna that a Maian craft was shot down near Area 51 and sends her to rescue a Maian protector named Elvis.
Because the President of the United States refuses to loan dataDyne the Pelagic II, the NSA sends a strike team to kill and replace him with a dataDyne-grown clone. The strike team invades the air base from which the Air Force One will depart. When Joanna foils this strike, the NSA and a group of cloaked Skedar take over the plane itself, which crashes after Joanna attempts to detach a craft attached to it. Having survived the crash, Joanna eliminates the President's clone and rescues the real President. Trent's incompetence angers Mr. Blonde, who kills him after disabling his cloaking device. DataDyne decides to hijack the Pelagic II and reach the ancient spacecraft. However, unbeknownst to dataDyne, the spacecraft contains a powerful weapon capable of destroying a planet and the Skedar intend to test it on Earth before using it against the Maian homeworld.
Joanna and Elvis follow dataDyne to the ancient spacecraft, where they find a reprogrammed Dr. Caroll cracking the weapon. Joanna replaces its current personality with a backup of the original, and the restored Dr. Carroll sets the weapon to self-destruct. As Carrington and Joanna prepare for a Presidential reception, the Skedar assault the Carrington Institute and capture Joanna. In space, aboard an alien spaceship on course to the Skedar homeworld, Joanna finds herself in a holding cell with Cassandra. Feeling that she has been used, Cassandra redeems herself by making a distraction and sacrificing herself, freeing Joanna and therefore giving herself a chance for revenge. With the help of Elvis, Joanna takes control of the spaceship and lands on the Skedar homeworld, where she ultimately defeats the Skedar leader, leaving the Skedar in disarray. The game ends with Elvis and Joanna leaving the planet just prior to an orbital bombardment from the Maian navy.