Eagles play an important role to the Assassin Brotherhood, in that many of the Order's outfits and weapons bear resemblance to eagles, and the birds swarm around View Points which the Assassins use to navigate their surroundings and perform Leaps of Faith from. In this regard, the Assassins dubbed the sixth sense that belonged to some of their members as "Eagle Vision".
Altaïr Ibn-La'Ahad was named after the brightest star in the Aquila constellation; he acknowledged this by designing the door to his library to resemble the constellation. Only by placing the Memory Seal keys in the etchings of the constellation would the door to his treasure room open.
Ezio Auditore da Firenze collected eagle feathers for his younger brother Petruccio. After his death, Ezio continued to collect feathers in memory of his brother, placing them in a box in his mother's room at the Villa Auditore; this tradition continued following the Siege of Monteriggioni, in a box at the Tiber Island headquarters.
The Kanien'kehá:ka revere eagles, and use their feathers in coming-of-age rites. During his coming-of-age, Ratonhnhaké:ton communed with Juno with a Crystal Ball, and the two spoke in the form of eagles. This conversation led Ratonhnhaké:ton to depart from his village and join the Assassins, though he would still collect feathers as a remembrance to his home. Achilles Davenport, Mentor of the Colonial Assassins, had a stuffed eagle preserved in his room on the Davenport Homestead.
- According to Jade Raymond, the series' eagle symbolism came about when the Assassin's Creed creative team realized the Assassin's predatory nature was akin to a bird-of-prey. Following this idea, they designed the character's robes to resemble an eagle and named him Altaïr.
- The franchise subsequently adopted a tradition of giving certain characters names associated with eagles (or other birds) such as Aquilus, Ezio, Haytham, Arno, Arbaaz, Orelov, Griffin and Callum.
- Desmond Miles possessed a tattoo on his left forearm, the top part of which resembled an eagle with its wings spread.
- The eagle's cry heard throughout the series is, in reality, the cry of a red-tailed hawk. 
- Connor's ship's name, Aquila, means "eagle" in Latin and Italian; also, the name Jackdaw is related to eagles, being a reference to the fable of the Eagle and the Jackdaw.
- An eagle appears at the beginning and the end of Assassin's Creed: Rogue, when Shay narrates the events of his life.
- The eagle motif is also present in the Assassin's Creed film, with Benedicto telling Aguilar de Nerha that "The spirit of the Eagle will watch over the future."
- In some cultures, Eagles are considered to be spiritual messengers between the gods and humanity.