A very old trope, in which left-handedness is a signal that that someone is the Villain or The Mole, or occasionally just susceptible to the Heel-Face Revolving Door. The word "sinister" actually is derived from the Latin word for left-handedness, and also related to the Bar Sinister which is a symbol of bastardy, and thus untrustworthiness.
This is also related to Evil Twin; twins do often have opposite dominant hands, and an Evil Twin will (almost invariably) be the left-handed one.
Additionally, in older times, left-handedness was often a sign of evil or trickery because of the fact that people would shake hands with their right, grabbing each other by the wrists, in order to show that they were friendly and didn't have weapons. A lefty could shake with their right hand, grasping the other person's dominant hand, and attack with their weapon in their left hand while the other was defenseless. Also, during the Middle Ages, towers were built so defenders would have no problem swinging their swords down staircases while attackers would have loads of problems swinging up. However, this only applied if both were right handed; if the attacker were left-handed, the two would be evenly matched.
In many cultures, before the invention of toilet paper and forks, the right hand was used for eating, greeting etc., while the left hand was used for wiping oneself and other less savory things.
Another related trope is for a character to lose his right hand, symbolizing his loss of a moral compass. This can presage a Face-Heel Turn by the Hero. It is also common for Mad Scientists to have a mechanical right arm. Alternatively, the left hand might become the repository of Sealed Evil in a Can. Compare Red Right Hand.
A subtrope is The Killer Was Left-Handed, where that trope there is actually evidence that the killer was left-handed. Examples of that trope should go there.
The opposite, when left-handedness is viewed favorably, is The Southpaw. Sometimes overlaps with Left-Handed Mirror. Also see I Am Not Left-Handed.
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Figures prominently in Read or Die. Really prominently - will ruin the main plot twist if you read the spoiler. Nancy is left-handed and is The Mole. Then to add to that she has an Evil Twin and they are differentiated only by their dominant hand. Inverted because the right handed Nancy is the Evil Twin. As an alternative interpertation, it is not inverted because left-handed Nancy betrayed Yomiko first, and then betrayed and killed her own lover (although he had betrayed her), while right handed Nancy was at least loyal to her man, making the left handed Nancy experience a Heel-Face Revolving Door.
Wrath of Fullmetal Alchemist may or may not be left handed, but he got the right one from someone else. The same could be said of Scar, though their alignments turn in opposite directions.
Gin from Detective Conan is left-handed. Also, there is a case Conan solves by realizing a man had been replaced by a duplicate because the fake was left handed and the original was not.
Higuchi in Death Note, though nobody mentions it. Light's ambidexterity probably counts as well.
One Piece: An interesting case with the Straw Hats, as none of them are evil, but a large number of them have something of importance on their left side. Luffy has his scar underneath his left eye. Nami's tatoo is on her left arm. Zoro's earrings are on his left ear, and although ambidextrous appears to prefer his left hand. Usopp always wears his wrist band on his left hand. Sanji's left eye is always covered. Chopper's antler plate is located on his left antler. Interestingly enough, the two Strawhats that finally break this pattern, Robin and Franky, are both introduced to the story as villains/antagonists to the Strawhats. Brook then completely subverts it, as his one asymmetrical trait is on the right side of his face.
Sanji also subverts this after the Time Skip, when his Peek-a-Bangs switch eyes to cover his right one instead.
Inverted by Nanoha, the left-handed heroine of the Lyrical Nanoha series, which is full of right-handed villains. Unless you take her fanonreputation into consideration.
In Naruto, Sasuke realizes that the Naruto in front of he and Sakura is an impostor because his knife pouch is on his left leg, implying left-handedness, while the real Naruto is right-handed.
Very subtle and not called into view, but all the members of Z-One's team, (Placido, Lucciano, Jose, Aporia, Paradox and Antimony/Bruno) all wore their Duel Disks on their right arm, and hence were all left-handed.
Inverted (thematically at least) in the Lone Wolf gamebooks: magic traditions inspired by the Forces of Light are referred to as "left-handed", while the Black Magic of the servants of God of DarknessNaar are referred to as "right-handed".
In The Bible, Ehud, one of the judges, actually managed to assassinate a king by hiding a miniature weapon on his right hip. Thus the weapon wasn't found, and he could still use it, being left-handed.
Arya from A Song of Ice and Fire. When her dominant hand is revealed to her fencing master, he reacts favorably because fighting left-handed will reverse her stance and movements, which will help confuse her opponents. However, her left-handedness might also be A Sinister Clue about her future, training in a cult of assassins.
Subverted with Jaime Lannister, who loses his right hand just before his Heel-Face Turn, away from the dark side.
In the novel The Sterkarm Handshake, the whole Sterkarm clan are lefties and are known for being backstabbers.
In Star Trek: The Next Generation, Data figures out he and Picard are trapped in a holodeck Enterprise when (the simulation of) Geordi catches a tool Data throws to him with his left hand, exposing a programming glitch Data was already aware of.
The logo of the Borg Collective looks like a grasping left hand.
In Graceland Briggs is left handed. This trope is ony invoked later in the first deason, with the big reveal.
Paranoia: "Left-handedness is one of the ten early warning signs of Commie mutant treason!"
Dyne, a side-villain in Final Fantasy VII, had his crippled LEFT arm replaced with a machine gun, and is distinctly portrayed as a serial killing lunatic. The reverse is true of Barret - even though he leads a terrorist organisation that blows up a power plant and kills hundreds of people, his RIGHT hand was the one replaced with a similar weapon. He turns out to be a nice, but misguided person who is a doting father and dedicated friend.
Sephiroth and his remnants are also lefties.
Kain Highwind, a party member from Final Fantasy IV, is left handed, which is shown by having weapon and shield slots in his inventory screen reversed. No points for guessing what he does, especially with a namelike that. Though he does get better... somewhat.
In God Hand, the titular artifact exists in a pair: left God Hand and right God Hand. The hero, Gene, got the right one to replace his severed arm. As for the left one, it's used by the Dragon Azel (who is the most evil in the group of villains).
Super Smash Bros.: Takes this to its Logical Extreme: The Master Hand is a giant, floating, apparently benign right hand who created the world that the characters find themselves in, and lives to be the ultimate challenge for heroes. The idea is that he is the hand of the child making the trophies fight, just like a kid in his room playing with action figures. His Evil Twin is the Crazy Hand, the left hand of the pair, who laughs maniacally upon his entrance and whose attacks and general movements are both bizarre and incredibly erratic. Whereas Master Hand is said to be a benign force and to represent the creative nature of a child, Crazy Hand is a destroyer, and supposedly represents that same child's more sadistic qualities.
As a comparison, in XD, Michael always makes a regular catch (at the Poke Spots, given that's where wild Pokemon appear from time to time) or calls out a Pokemon with his right hand. He only uses his left when he needs to make a Snag.
In one "Treehouse of Horror" episode of The Simpsons, left-handed Bart turns out to be the evil twin of Hugo Simpson. Bart's left-handedness is never brought up in the episode though.
There's no indication as to whether either Bart or Hugo is left- or right-handed; however, Bart was the left-sided (or Sinister) twin.
Francine from American Dad! was raised to think lefties were evil.
In one episode of The Batman, Mirror Master creates evil duplicates of Batman, Robin, and Flash. Batman quickly notes that the duplicates are left-handed.
Examples of the related tropes:
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The left hand of the titular character of Hell Teacher Nube is actually a sealed Oni, Baki, which replaces the left hand he lost fighting said Oni. Most of the time, it looks like his regular hand wearing a black leather glove; when his students are threatened by spectral horrors, he will reveal it as a monstrously large, dark red hand with visible tendons and black claws. However, he constantly seeks a way to seal it permanently, as only the influence of his childhood teacher prevents the Oni from taking over his entire body.
The lead characters of Parasyte are a young man named Shinichi and the alien parasite that has possessed/replaced his right hand. Their relationship is one of the main foci of the series, especially re: Shinichi losing part of his humanity. As early as the first volume, the Love Interest intuits there's something weird about his right hand and switches around him to hold his left instead.
The original English translation was flipped and 'Migi' was named 'Lefty' instead.
Edward Elric of Fullmetal Alchemist lost his right arm and his brother lost his entire body performing forbidden alchemy, which put him on a self-imposed Redemption Quest to restore his brother's real body. At the end of the story, he has his right arm and his brother back, but Edward had to give up his ability to perform alchemy.
Lex Luthor also had a mechanical right hand for a while; he lost the original to Kryptonite poisoning. Naturally, he took Rotwang's role in the ElseworldSuperman's Metropolis.
In Star Wars: Attack of the Clones Anakin loses his right arm- symbolism of a move towards the Dark Side? Luke then loses his right hand in the same way- right before The Reveal that Luke, I Am Your Father, and in Return of the Jedi Luke breaks free from his Freak Out after he cuts off his father's right hand and realizes that both of them have mechanical right hands.
Inverted in A Song of Ice and Fire. When Jaime Lannister loses his right hand, his moral compass actually starts working better. This is mostly because without his sword-hand, he's forced to think things through, when before he could just hack his way through any obstacle.
The main villain of Final Fantasy IV, Golbez, is left-handed. However, you only learn this when he's freed from brainwashing (done by the real villain) and becomes a protagonist in the sequel. He wields weapons in his left and shields in his right. Similarly, Kain is left-handed, and ends up brainwashed to fight for the villain.
Notably inverted with a lot of the characters Shigeru Miyamoto creates. Mario and Link are both left-handed (though the default latter has changed with the sword swinging of the Wii titles), and Miyamoto himself is ambidextrous, preferring his left hand.