According to Chris Priestly, Tali'Zorah nar Rayya was almost not included in the game as a full squadmate. Writer Patrick Weekes was able to persuade the development team to include her in the game as a full squadmate.

In 2021, former Bioware cinematic designer Richard Boisvert posted an easter egg to Reddit that he believed had never been found before. In the Mars mission, if Commander Shepherd walks a specific route through the solar panels at the start, a Mars rover will roll up to Shepherd and nod.

If the player romances Tali'Zorah, a photo of her will appear on a drawer next to Commander Shephard's bed. The photo itself is actually an altered stock photo with a model named Hammasa Kohistani. This quickly drew controversy not long after the game was released, alongside with other strong criticisms of the game.

The Illusive Man was first intended to turn into a Reaper creature in Mass Effect 3. This plan was later scrapped as the developers wanted to give players the satisfaction of fighting a character they knew rather than a random creature, highlighting the fact that the Illusive Man's weapon is his intelligence, not his physical strength.

The pilot, Joker, references the book and film The Hunt For Red October by saying, "Stealth drive engaged. Only way they'll detect us is if you all start singing the Russian national anthem." In the book and film, the eponymous submarine, thought completely undetectable by traditional means, is discovered by American sub sonar when its crew joyously sings the Russian national anthem.

Should Ashley have survived Virmire in the first game, after the "Shut down the Geth server" mission, the player can find a hungover Ashley lying on the floor after a night of hard drinking.

If Wreav is the Urdnot leader during the mission Priority: Tuchanka, he will urge Shepard to "fight like a Krogan". This is a reference to the chorus of the Mass Effect fan song 'Commander Shepard'.

If you chose Kaidan Alenko as the living squad member (leaving Ashley Williams to die in Mass Effect 1) you can see Ashley's ghost on the Citadel in a cutscene. If you chose Ashley in Mass Effect 1, nobody is standing in her place.