There's a mission in which you have to deliver a letter to a farmer. Upon reading the letter, you learn that the farmer is charged with attacking neighbors with swords. Additionally, it states the farmer has "committed crimes against God and nature" and says "if you are not aware of the charges against you, read Leviticus 18:23." Leviticus 18:23 is a verse of the Bible about the wrongs of sexual intercourse with animals.

In a dungeon of the game, if you pull a lever and stare off into the water for about 30 seconds, a monstrous squid with glowing eyes will slowly swim by. If you pull it and wait again, the squid will raise a tentacle to attack Ezio.

When Sequence 3 starts and you are escorting Maria and Claudia to the villa, you will get ambushed by Pazzi and his men. You will be rescued by your Uncle Mario. When you first talk to him, he says "Don't you recognize me? Its-a me, Mario!" - a popular quote from the Nintendo hero of the same name.

There is an Easter egg that can only be seen with Eagle Vision in the 'Bonfire of the Vanities' DLC, and only at a certain time. After meeting up with Niccolo, go to the Palazzo Auditore and enter the building. Engage Eagle Vision and you will see the dead family members of Ezio.

Due to a developer oversight, it's possible to use a broom to slit an enemy's throat.

According to the game's manual, not having the ability to swim in Assassin's Creed 1 was a 'bug in the Animus' preventing people from doing so, not Altair's inability.

In an interview with creative lead Patrice Desilets, he confirmed that the DLC was originally planned to be part of the main game.