- An announcement that plays in the Spaceflight Terminal states that "over 5,000 people currently live and work" on Sevastopol. However, The Art of Alien: Isolation states that the station has a maximum capacity of around 3,000 inhabitants, with only 500 on board at the time the game's events begin.
- Just before Axel is killed, the Xenomorph's drool lands on his shoulder, implying the creature is above him. However, when it attacks, it takes him from the side before dragging him through a vent in the wall nearby.
- When the player first enters Seegson Communications, they witness a Working Joe murdering Hughes by bludgeoning his head against a wall, splattering the surface with his blood, more of which pools around his corpse on the floor. When Ripley returns to the area much later in the game, Hughes' corpse and the blood will be gone. While it is conceivable his corpse may have been taken away, it seems unlikely anyone would also take the time to clear up the blood.
- The Pilot's chamber aboard the derelict ship on LV-426 is far larger than it was in Alien. The walls are also further from the central platform and feature openings that were not present in the film, most obviously the one through which Marlow passes to deactivate the beacon.
- The melted hole that Kane used to access the derelict's cargo hold is located behind the Pilot's chair in the game. In the film, it was located to the chair's left.
- The Eggs in the derelict's cargo hold are grouped into large recesses separated by raised, walkway-like ridges. In the game, each of these recesses is covered in blue mist, but in the film, only one of the recesses is filled with this mist (although it could be argued this was due to budget/effects limitations in the film).
- Despite the fact the Anesidora crew apparently explore the exact same area of the cargo hold as Kane, there is no sign of the single open Egg that should remain from when he was attacked.
- Sinclair signs his penultimate archive log with the name Jake, yet his ID tag, found nearby, gives his first initial as "F".
- Whenever a Seegson Security officer is headbitten by a Xenomorph in-game, their helmet and visor will remain intact.
- The flamethrower that Amanda Ripley uses aboard Sevastopol is almost identical in design to the Flame Thrower used by the crew of the Nostromo, yet the latter was supposedly a custom tool fabricated by Parker specifically to deal with the Alien; it seems highly unlikely the two would be so similar in appearance.
- The content of archive log 078 implies it should be an audio log; for instance, the sender asks the recipient, "Can you hear me?" However, it is actually a simple text log; in this context, asking the reader if they can "hear" the message makes no sense.
- The maps for the Lorenz SysTech Spire upper transit and Solomons Habitation Tower habitation decks in Amanda's journal during mission 17 are incorrectly labelled, the titles having been swapped between the two.
- Parker's character model lacks the beard he had in the film.
- Dallas' jacket lacks the laces on the sleeves that can be seen in the film, looking more like Parker's jacket.
- Lambert's corpse is in a different position to that of the original movie.
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