Freudian Threat

"Salario: Why, I am sure, if he forfeit, thou wilt not take his flesh: what's that good for? Shylock: To bait fish withal."

- William Shakespeare, The Merchant of Venice

A threat intended to make someone sexually uncomfortable; more often than not, a character threatens (sometimes subtly) to castrate someone.

Comic Books
"Dwight: You ever so much as talk to Shellie again -- you even think her name -- and I'll cut you in ways that'll make you useless to a woman."
 * During the opening scene of the Sin City story "The Big Fat Kill," Dwight McCarthy, having dropped in on an asshole by the name of Jackie-Boy who's been abusing his ex, issues this vicious threat:

Film

 * Fatal Instinct. In the Backstory, Max Shady threatened to cut off his lawyer Ned Ravine's penis after Ravine lost Shady's case.
 * Men in Black: "I want you on the next transport off this rock or I'm going to shoot you where it don't grow back."
 * In The Three Musketeers 1993 when Cardinal Richelieu starts making advances on Lady DeWinter she pulls a knife and holds it to his crotch. Richelieu warns that he can have her executed with "a snap of [his] fingers" to which she replies, "And with a flick of my wrist I could change your religion"; he backs off.
 * Fight Club. The threat to cut off someone's balls happens a few times.
 * Nine to Five. Finally losing her temper at her Bad Boss, Doralee (Dolly Parton) threatens to "get that gun of mine, and turn you from a rooster to a hen in one shot!"
 * A Clockwork Orange. Mr. Deltoid, a truancy officer, grabs protagonist Alex's crotch while warning him of the dangers of his skipping school.
 * However, his extremely creepy actions throughout this scene suggest he did this more because he's a pervert than because he was trying to frighten Alex.
 * And who can forget The Big Lebowski and the three German nihilists who corner the Dude in his Candlelit Bath and threaten to cut off his "johnson?"
 * Jennifer's Body had a hilarious one in the unrated cut where Jonas' father threatens to cut off his son's killer's nutsack and nail it to his door.

Literature

 * Unsurprisingly, the Discworld has a few examples:
 * Cohen the Barbarian's explanation of psychological warfare in Interesting Times: "It's where you bang your shield all night before the fight so's the enemy can't get any sleep and you sing, 'We're gonna cut yer tonkers off!' and stuff like that."
 * In The Fifth Elephant, Commander Vimes corners a suspect who tells him to "look down" at the knife he's holding to Vimes' stomach. Vimes' reply? "Look down further."
 * In Yellow Blue Tibia, the protagonist is threatened by a cop, who is using a tape recorder to record everything but the threats, and who goes on and on about cutting that off and, while any hospital can transfuse blood, "there is no hospital in the world that can transfuse you a new set of balls!" The protagonist (inwardly) chuckles at this one-note attempt at intimidation, made worse when.

Professional Wrestling

 * Manager Yamaguchi-san threatened wrestling pornstar Val Venis, on the occasion of Venis making time with Yamaguchi's wife: "I CHOPPY-CHOPPY YOUR PEE-PEE!!"

Theater

 * The legendary exchange between Salario and Shylock in The Merchant of Venice, quoted above.

Video Games
"Elias Kelham: Are we done here? Because I got people to see. (Shepard slams his/her forearm against the guy's throat) Shepard: I'm done being patient. Give me a name, or I'll cut your balls off and sell them to a krogan!"
 * During an interrogation during Thane's loyalty mission in Mass Effect 2, a Renegade Shepard can choose to rough up the suspect and wrap things up like so:


 * In No More Heroes, Sylvia says to Travis: "If I ever hear you mumble another woman's name in your sleep, you'll wake up the next morning with your joystick missing."
 * A well-known part in Final Fantasy 7 where Cloud, Tifa and Aeris threaten the resident pimp Don Corneo while likewise interrogating him by telling him they'll "cut", "rip" and "smash them" in turn.

Web Comics

 * Berthold the Wolf from The Last Days of Foxhound frequently makes more-or-less subtle threats to bite off other character's dicks if they don't do as they're told. (Since the only female member of the team is his mistress, Sniper Wolf, it's a convenient sort of threat.) At one point, Liquid comments on how often he uses that specific threat, and he basically just shrugs and goes "Hey, it works..."

Western Animation

 * In X-Men: Evolution, Rogue catches Spyke videotaping her without her permission. She tells him that, if she sees herself in his video when he presents it to their class, "They're going to call [him] Spyke-less."
 * On The Boondocks, in Grandad's tale of "The Story Of Catcher Freeman", after the other slaves trick Masta Colonel into condensing his efforts at intimidation onto solely his Butt Monkey house slave Tobias, Masta Colonel states that if their work isn't done when he next comes out, "I'm cutting both [Tobias'] balls off."