F.O.W.L. antagonizes Darkwing Duck on different occasions:
- The High Command - They are the head agents of F.O.W.L. It seems that throughout the entire series they never left their certain area (the area they were was possibly F.O.W.L. HQ). Also, they were always hidden in the shadows, and their true names were never mentioned during the Darkwing Duck series run at all. The ones that usually talked were the one with a big head and fangs and the one with a hunched back, though not always with the same voices (in "Heavy Mental", the large-headed one is a woman). The one wearing the oversized hat never talked. It also appears that they were anthropomorphic birds, most likely vultures. All members of F.O.W.L. are shown to fear them, as they appear to be prone to violently dispose of those who upset them, including Steelbeak's predecessor, who was put through six trash compactors.
- Steelbeak - Acting like a 1920s gangster, Steelbeak is a rooster who is the top agent of F.O.W.L. He gets his name from the metal beak he wears in place of his original one which was apparently lost sometime before the events of the show, though it is not revealed how. This trademark beak also serves as a weapon because it can bite through almost anything.
- Ammonia Pine - A former cleaning lady who worked at a research lab, Ammonia accidentally inhaled the fumes of an experimental bathroom disinfectant, which had a strange effect on her and turned her into a cleaning-obsessed villain. She was later recruited by F.O.W.L. for their dastardly plans. She hates dirt and is even afraid of it. She has a sister named Ample Grime, also a F.O.W.L. agent.
- Ample Grime - Ample is Ammonia's messy, dirt-loving sister. No origin story is given for why she loves messes so much. She hates and fears cleanliness as much as Ammonia hates dirt, which proves to be a problem when the two are required to team up.
- Major Synapse - The typical R. Lee Ermey-type military man. He once sent an Eggmen military unit into an active volcano so it could be "captured." He steals S.H.U.S.H.'s Norma Ray, using it to turn his useless hippie underlings into superpowered malcontents. He later used it on himself, turning into a giant head with a giant brain that had major telepathic abilities until his own stupidity caused his underlings Hotshot and Flygirl to be flattened by a cartoon anvil which they are last seen crawling away under. His own superbrain explodes when Darkwing Duck, Launchpad, and Gosalyn overloaded him with questions.
- Hotshot - One of Major Synapse's hippie underlings changed by the Norma Ray. He controls both fire and ice.
- Flygirl - One of Major Synapse's hippie underlings changed by the Norma Ray. She can move things with her mind and can also fly.
- Eggmen - F.O.W.L. foot-soldiers, instantly recognizable by their white, egg-shaped helmets.
- Wolfduck - Wolfduck was created solely for the Capcom video game. He does not appear in the TV series or the comic book. Because of this, not much is known about him or his backstory. Presumably, he was bitten by another wolfduck at some point in time and goes berserk when transformed.
- Mr. Wonderful - A parody of the Fantastic Four's Mr. Fantastic, seen in the Disney Adventures comic story "Super Union Blues".
- Razor Quill - Appears only in the Disney Adventures comic story "Gosalyn Alone".
In this version of F.O.W.L., they are a secret evil organization that watches the world from its shadow. It was first referenced in "From the Confidential Casefiles of Agent 22!".
After the events of "Moonvasion!", the society could no longer let Clan McDuck keep running loose as they almost cost them the world (due to Della's big mouth disillusioning the moon's inhabitants of their belief the moon was a planet), and therefore assures that they will give them the last adventures they will ever have.
In "Challenge of the Senior Junior Woodchucks!", F.O.W.L. spies on Clan McDuck through a robot mosquito and learns they had acquired the lost journal of Isabella Finch.
Using one of the missing mysteries known as the Third Eye Diamond, F.O.W.L.'s top agents intended to use it in an intella-ray to wither Scrooge's mental acuity to a point where he would be less than harmless in "Double-O-Duck in You Only Crash Twice!". The plan quickly goes awry and one of the Directors, Bradford Buzzard, ends up having to clean up the mess after telling said operatives to avoid arousing the ducks suspicions.
Despite F.O.W.L.'s attempts to stay hidden, they were finally exposed in "Let's Get Dangerous!" when Bradford called for a heli-evac, which had the F.O.W.L. logo on it.
In "The First Adventure!", the origins of F.O.W.L. are detailed: Bradford was once an accountant working at S.H.U.S.H. who disliked of how much money the organization wasted. He thus proposed they just take over the world to reign in its chaos while making a profit in the process, but was laughed off by the leadership. This prompted him to co-found F.O.W.L. alongside Black Heron. Noteworthy was that Bradford originally intended to make the organization's name O.W.L. (the Organization of World Larceny) but Heron added the F (for Fiendish), which she thought sounded cooler.
- Bradford Buzzard - Originally, Bradford, Bentley, and Buford Buzzard are presented as Scrooge's trusted advisors, the Board of Directors are three vultures that manage McDuck Enterprises finances, meeting with Scrooge, and at one point, Louie, on occasion to discuss his spending and make cuts, being partially responsible for Della being stranded on the moon after cutting Scrooge's funding for the search. At the end of season 2, they are revealed to be leaders of F.O.W.L., asserting that they will give clan McDuck their last adventure. Bradford is one of the organization's founders.
- Gandra Dee - Initially portrayed as a rebel scientist and Fenton Crackshell-Cabrera's love interest, she is later revealed to be working with F.O.W.L. at the end of season 2. However, she is later revealed to only be using F.O.W.L’s processors to help Fenton create a digital workspace where she can experiment without fear of ever hurting anyone, and attempts to quit F.O.W.L after it’s completion, only to be captured instead, but was later freed during the final battle.
- Phantom Blot - An agent of F.O.W.L. who wore the costume of Funso, the mascot of family restaurant Funso's Fun Zone. At the end of season 2, he removed his costume's head to reveal his true form underneath. He seems to only be a member for resources to kill Magica.
- John D. Rockerduck - Originally an Old West robber baron, he crossed paths with Scrooge and Goldie O'Gilt before losing a giant gold nugget he had stolen from the town of Gumption to the pair. He is later affiliated with F.O.W.L., frozen in cryogenic suspended animation. He was later thawed from his suspended animation and rejuvenated his youth after drinking from the Fountain of the Foreverglades.
- Jeeves - Rockerduck's enforcer who was bribed by Scrooge in order to make off with the giant gold nugget he had stolen. He later assists the frozen Rockerduck, bearing a new stitched together look.
- Steelbeak - A F.O.W.L. operative with a metal beak (hence his name), Steelbeak was shown at the end of season 2. Unlike his previous incarnation, who was suave and smart, this Steelbeak is a moron.
- Black Heron - A chemist turned agent of F.O.W.L. and an old enemy from Agent 22's time as a S.H.U.S.H. agent who returns to exact revenge and get her hands on the recipe for gummiberry juice. She also created the Rescue Rangers by testing an Intelligence Ray on a group of lab rodents, who subsequently began working against F.O.W.L. Black Heron is one of the organization's founders.
- Don Karnage - Replacement for Gandra Dee following her defection.
- Eggheads - F.O.W.L. foot-soldiers. The new name for the foot-soldiers reflects that there are both male and female soldiers employed. The one named Pepper is Phantom Blot's partner.
- The book Darkwing Duck: The Darkest Night incorrectly shows Taurus Bulba as the leader of F.O.W.L.. Although he is a member of F.O.W.L. in the DuckTales reboot, he revolts against them in the end.
- F.O.W.L. is a parody of S.P.E.C.T.R.E. (SPecial Executive for Counterintelligence, Terrorism, Revenge, and Extortion), a global terrorist- and criminal organization from the James Bond franchise.