The Ark Brotherhood is a group of scientists and researchers located underneath The University of Science and Technology of Rolla city. Its founders consisted of surviving teachers, professors and students that have hidden below the school in emergency fallout bunkers. There they remain underground with the supplies that they have and by the year 2100 the survivors and their descendants would found the Ark Brotherhood, originally called "The Brotherhood", an organization dedicated to technology and developing more sources of energy.

For decades they would remain isolated from the rest of the world as they performed experiments and tests while developing all new sort of technology that would aid them in their survival. In the year 2168, with one of their experiments, they would discover a new type of energy that can be used as a weapon which they dubbed "Ark". It would be capable of shooting bolts of arc based energy onto an enemy and has a chance on paralyzing them.

Out of all of the factions, they are the most advanced and well hidden. No one besides the Enclave know of their existence.

They can be found late into the game when either working with the Enclave or with the Midwestern Brotherhood of Steel.