While it's never actually shown, in Portal 2, during the section where Wheatley steers your Relaxation Chamber through the Relaxation Center, you can see shipping labels on other rooms, which (among other things) label the occupant of that chamber as either an adult or a child. This implies that there were children in the Relaxation Center, meaning GLaDOS had probably used children as test subjects in the past. Considering the deadliness of her tests, children probably also died horrific deaths at her metaphorical hands.
Apollo Justice: Ace Attorney: Kristoph Gavin attempted to murder a 12-year-old agoraphobic girl by giving her poisoned nail polish, since he knew she had a habit of biting her nails when under pressure.
Phoenix Wright: Ace Attorney – Spirit of Justice: Queen Ga'ran was willing to let Apollo die in a fire she started when he was an infant, while killing his father in the process. At one point she even implies she'd be willing to have her 14-year-old niece killed.
In Alice: Madness Returns, the Big Bad is a pedophile and sexual predator who has killed children, plain and simple. This trope is his most defining "evil" trait.
Metal Gear uses this rather a lot, either to demonstrate the generalized war setting or as Kick the Dog for the villains:
Solid Snake (or rather, the player) in Metal Gear 2 is actually given the option of murdering orphaned war children in Zanzibar Land, and despite being raised in a soldier nation, apparently they don't have orders to attack Snake or sound an alert (they just seem to just give advice and talk to him), although it does result in his health going down.
In the (albeit non-canon) radio drama for Metal Gear Solid, Solid Snake and Mei Ling argue about whether there is a significant difference to killing child soldiers and regular soldiers. Solid Snake mentions that he doesn't see a difference, which indicates that he is not above killing child soldiers.
In Metal Gear Solid 2, Solidus Snake's depth of evil is characterized with how he used to be a warlord with an army exclusively made up of child soldiers (called the Small Boy Unit), killing their parents and then sending them off to die in the Liberian civil war. In the same game, the evil of the Patriots is highlighted by their kidnap of Olga's child, who they use purely to keep Olga under control.
Metal Gear Ac!d concerns two outrageously evil Mad Scientist characters who kidnapped several hundred children and forced them to fight each other to the death to find out which one had the greatest potential. The one who won was then made into the operating system for an extremely powerful new form of Metal Gear. The trauma of performing this experiment was so great that one became practically a Card-Carrying Villain to deal with it and the other (who had taken on the work under a Code Name) repressed all his memories of it and went off to go and live in Alaska, returning to his previous identity of Solid Snake. Or so it seems.
The main villain in Metal Gear Solid: Portable Ops has a child soldier who looks quite old but, if you look at the numbers, cannot be older than twelve - Null, a soldier who is kept in total sensory deprivation and essentially tortured into repressing all of his memories after every combat with the idea that this will make him a 'Perfect Soldier'. The villain's open exploitation of and the physical violence he uses towards Null to control him is one of the many reasons he is profoundly unsympathetic. Null was also a child soldier earlier in life, as well.
Return to Krondor: Played straight. In the first chapter, a ten year old girl who is a thief and an orphan living in the streets truly does not want to go to The Order of the Yellow Shield, a group that owns an orphanage. That is because men who pretend to be members of The Order of the Yellow Shield lure kids like her to a sweatshop. At this sweatshop, they work the kids hard, hurt them, lock them up in cages, as well as giving them food that have live rats and squirmy things in it. She also says about how the bad children (i.e. kids who refuse to work or try to run away) just disappear and never come back. Investigating the sweatshop reveals that everything she said is true. You will find a cage with kids locked in it, and depending on your decisions, you will find the bloody corpse of a child in one of the boxes next to the entrance door. James will be very unhappy with that discovery and refer to the owner of the sweatshop as a "child-killer". A camp of goblins sacrificed a boy, cutting him in two, and they were going to do the same to baby twin girls. Vampires killed and converted kids as well as adults to vampires. Ghouls are explicitly said to feast on human flesh - and that would include children. There is also a priest who is devoted to Sung the Pure named Father Roweland who is trying to help children recover from a fatal disease, but he causes the fatal "disease". He actually killed a little boy with evil spells, and was going to do the same to a little girl with an evil amulet magically disguised as a good amulet, as well as evil spells. A woodcutter, his wife and child disappear, but the woodcutter and his wife (not really his wife) sacrificed their child (not really his child) to try to power up a Nightstone. The Nightstone is found on a small skeleton, which could very well be the child's remains. Bear also used explosives to set an orphanage on fire while escaping Krondor - with the kids still in it.
A side mission of Drakengard features Anti-Hero Caim slaughtering child-soldiers with about as much gusto as he slaughters everything else, i.e. a lot. The children run away and scream for their mothers as you cut them down, while Leonard and Red calls you a monster for it. And then there's that child-eating Elf cannibal he travels around with, and Leonard was originally a pedophile in the Japanese version... Yeah, Drakengard doesn't like children a whole lot.
And in Alternate Continuity sequel NieR, some of the shades that Nier slaughters are actually sentient children who cannot communicate with the "humans", particularly Kalil, the "boss" of the Junk Heap area.
The plot behind Heavy Rain centers around a serial killer that targets boys from the ages of 8-13. It makes replaying the Suicide Baby chapter much worse once you know who the killer is...
Anybody in the Kingdom Hearts universe will have no trouble with fighting the main characters, most of whom are teenagersnote Terra and (possibly) Aqua being the only playable characters over 17, so they have to take all their opponents seriously, so it's all fair play. Master Xehanort even manages to kill one of them (Sora) and steal the body of another (Terra), though the former gets revived.
Dragon Age: Origins is a rare game which lets a player character do this, as you can chose to murder Arl Eamon's son rather than try and help him. You might argue that it's a mercy-killing, but you're still killing a child.
The Human Noble origin has your character find their sister in-law and young nephew dead during the attack on the castle. By this point, it's clear the attackers don't care about hostages.
And we have this little gem right before the attack:
Mars and his soldiers in Tales of Phantasia. They kill everyone in Toltus, including Chester's little sister and a random girl whose corpse you can examine.
FunFrock in Little Big Adventure 2 initially uses kidnapped children in order to force the wizards to co-operate with his scheme. When Twinsen invades his lair, he decides to drop the children into a volcano in order to make Twinsen cross the Despair Event Horizon.
With all the Little Sisters running around pumped full of sweet, sweet ADAM, most Splicers will try to kill them if given the chance. Good thing the Big Daddies are there to protect them.
Also, the heartless bastard who put the girls in this situation, Doctor Suchong, is later killed by a Big Daddy after he slaps one of the Little Sisters who was irritating him.
BioShock Infinite: Daisy Fitzroy falls into this trope when she attempts to murder a young Founder child because, as she says, "the only way to stop them from growing is to pull them up from the roots!" This is enough to stir Elizabeth into impaling Fitzroy with a pair of scissors. However, Burial at Sea: Episode 2 reveals that Daisy was ordered to do so by the Luteces in order to strengthen Elizabeth's resolve by getting her to kill in order to set forth the events of Comstock's downfall and that she was actually horrified at the idea of killing Fink's son.
In The Elder Scrolls V: Skyrim the vampire Alva forces her thrall Laelette to murder a man's wife and young daughter as part of her plan to seduce him. Partially subverted by the fact that Laelette felt guilty about the whole situation and attempted turning the girl into a vampire and resurrecting her when that failed, but Alva had no qualms about killing the innocent.
The first two Fallouts make you a Childkiller if you killed a childnote Well, at least two, which would cause almost everyone, even other renounced scumbags and lowlifes, to hate you. At least the children were scripted to flee when accidentally hit, so they rarely got killed by NPCs. In Fallout 2, once you've progressed through the game long enough in an attempt to go to another settlement, you'll get an encounter that shows an Enclave patrol coldly killing a small family, including an only child, which serves as an ominous Foreshadowing to their existence, who have no qualms in exterminating any being that isn't them or wasn't born in a Vault, and yes, that includes children. The 3D ones had invincible children (unless you use some mods...there is even a very famous one for Fallout 3 which allowed eating a baby in exchange for the benefit of a perk which give you rads immunity, you can see this marvelous sickness here)
In Fallout 3, the Lone Wanderer can, however, enslave selected children NPCs and cause the implied deaths of two others, by blowing up a town with a nuke.
Fallout: New Vegas has two slaver traders making a note not to do any more business with the raider leader Cook Cook, as he burned a young boy they had sold to him to death. The Courier can also ask a prison rioter who goes by the nickname Scrambler what he was in for. He responds that he doesn't really care enough to keep track of his crimes, but he vaguely remembers something about killing a lot of people, and that some of them probably were kids.
According to Ranger Andy, Caesar's Legion considers using child suicide bombers to be a legitimate strategy since everyone lowers their guard with kids.
In Fallout 4 there are children found in both the Prydwen and the Institute, and you're forced to blow up one or both of them depending on who you side with. Also, when blowing up the Institute, you can leave the synth version of Shaun to burn, which gets you some harsh comments from your faction's tech specialist.
Final Fantasy XIII has Hope Estheim - the youngest, most physically weak character in the game. That doesn't matter to anyone; he's a Pulse l'Cie, and therefore must die.
Before this, he and a town were put on a train, and promptly shot at by the military when they tried to escape.
All the main protagonists in the MOTHER series are children (so obviously, plenty of the enemies that battle them will be adults), but perhaps the most notable specific example of this trope is in EarthBound when a bunch of policemen beat up the 13-year-old Ness just because he wants to leave his hometown.
MOTHER 3: Not only does Porky try to kill the 12-year-old Lucas on a number of occasions, he also takes his twin brother, mind controls him, makes him a cyborg, and ultimately is the cause of his death. Just so you know, this game is rated Japan's equivalent of E.
The Legend of Zelda: The Wind Waker has Ganondorf hitting Link, who is around 12 years old in this game, multiple times until he loses consciousness and has his sword knocked from his hand. Later, during the final battle, Ganondorf uses some very brutal and painful-looking sword moves against Link.
The Witcher 3 offers a vast amount of morally repugnant characters, but one ranking relatively high on the list would be Jonna. A local man named Lothar goes to Geralt for help lifting a curse from his young son that was slowly killing him. After doing some investigating, Geralt learns that Jonna, the local herbalist, cursed Lothar's son after Lothar refused to continue cheating on his wife with her in an attempt to win him back. Geralt can either transfer the curse to Jonna, killing her, or convince Lothar to go back to her so she would lift it herself.
Early on in Pokémon XD: Gale of Darkness, a thug working with Cipher called Zook threatens Michael and his younger sister with a Shadow Zangoose. And in the end Grandmaster Greevil's son Ardos attempts to talk his father into blowing up the base with Michael still on it after his defeat. Needless to say, it doesn't work.
In Pokémon Sun and Moon, after defeating her a second time, Lusamine, having fused herself with the Ultra Beast Nihilego, completely snaps and proceeds to try and physically attack her 11 year old daughter (and the player). Thankfully, Lillie's able to stop her with Solgaleo/Lunala's help.
The Shroobs and their army in Mario & Luigi: Partners in Time seem perfectly willing to kill Baby Mario and Luigi after beating their adult selves. Kamek too, who's also technically guilty of this in Yoshi's Island and its sequels (he starts by attacking the Delivery Stork in the intro, then tries to get rid of the Mario Bros. by any means possible afterwards).
Mario and Luigi also have no problems with killing a baby and are seen doing it 5 times in the Super Mario Galaxy games (2 Dino Piranhas, 2 Fiery Dino Piranhas and 1 Peewee Piranha) and in these cases (unlike the cases where the infant survives like Baby Bowser, Bowser Jr. and Jr. Troopa) they aren't being evil nor mean (though they still try to kill the Mario Bros).
Metro: Last Light has its fair share of children dying or dead. Most notable examples are the bombs that forced people down into the Metro, as well as the synthetic Ebola epidemic that Korbut released on the neutral stations that takes out every man, woman and child there- not to mention the executioner mop-up crews that are sent in to set the place ablaze and kill absolutely everybody.
Baldur's Gate has the gnome Jeb, who enjoys murdering street children. Unfortunately, you have to make allies with him in the first game in order to escape from prison; in the second game, however, you encounter him again, and can give him what he deserves.
Dr. Ivo Robotnik, the Big Bad of Sonic the Hedgehog. He not only shoves little animals into robots, but his worst enemies also happen to be children. Even not counting Sonic himself (who is often placed at 15, though he's implied to be younger in the first few games), he is fighting 8-year olds and 12-year olds on occasion (heck, Cream from the Sonic Advance Trilogy is only 6). Justified that they are children with Superhuman abilities, and rather mature-acting children at that, but still.
On the Game Gear, Eggman truly pumps this up a notch in Sonic the Hedgehog 2. It is implied that Tails dies at Robotnik's hands if you fail to collect all the emeralds before Scrambled Egg Zone.
Telltale's Game of Thrones: Ramsay Snow shows the Forresters just what an irredeemably evil bastard he is by gleefully stabbing the young Lord Ethan in the throat in front of them. Ethan was only around thirteen or fourteen at the time. Ramsay also takes the seven year old Ryon as a hostage and tells his soldiers to kill him if anyone tries to intervene.
Skull Girls: Black Dahlia, the Medici mafia's top assassin did not even hesitate to put a bullet through the head of fourteen year old Sienna Continello and actually taunted the poor girl's mother while doing it. She also threatens to kill the also fourteen year old Peacock in the latter's ending, but in that case it is more justified as Peacockhad been slaughtering her way through the Medici stronghold up to this point.
Season Three has Badger, who shows himself to have no problem with murdering children in cold blood including Javier's niece Mariana, which the majority of players agree makes bashing his skull until the remains are unrecognizable in Episode 3 all the more cathartic.
Five Nights at Freddy's: If you read the newspapers scattered throughout the game, you'll find out that some guy had the decency to kill not one, but five children. Minigames in Five Nights at Freddy's 2 suggest the death toll may be even higher than that. Five Nights at Freddy's 3 confirms a body count of at least five or six, and possibly up to eleven, kids. He's never even seen outside of minigames (where he's seen as an Atari-esque purple man) and in 3, where his corpse is seen inside Springtrap, much less named.
Five Nights at Freddy's 4 has the Nightmare animatronics who are terrorizing a literal child. Granted, they are literal nightmares, but still. There's also the Big Brother Bully of the child protagonist, who loves torturing his brother by exploiting his fear of the animatronics and was the direct cause of the accident that crushed his brother's head. Though that did lead to his My God, What Have I Done? moment.
In Far Cry 4, we eventually learn that Pagan Min's hatred of the rebels is due to them murdering his infant daughter.
In Overwatch's cinematic trailer, Widowmaker shows no qualms about trying to shoot two kids.
Vivi and Eiko from Final Fantasy IX. The former is a child physically and mentally, but since he's a black mage "doll", adult workers in Dali promptly kidnap him and stuff him inside a box. In the latter case, Alexandrian jesters kidnaps her and perform a ritual to steal her spells. Due to an RPG format, both are defaultly subjected to physical and magic attacks, especially those from Kuja's minions.
If you play the game as violently as possible and kill absolutely everything you can kill, you find yourself face to face with a major character who knows everything you've done, by which point you pretty much deserve all the hurt he can throw at you:
While we're on the subject, every character who isn't Toriel (who won't purposefully land a killing blow), Papyrus (who only weakens you to capture, not kill), and Alphys (who doesn't fight you until she becomes a lost soul), as well as the shopkeepers and a couple of other NPCs, attacks the player with the intent to kill on their own at some point. Justified because they need your soul to leave the Underground, along with not exactly being on friendly terms with Humanity to begin with (though some are rather reluctant to kill you anyways).
Flowey stands out in this regard. He doesn't try to kill you at the intro because of the same reason as the other monsters. He just does it for shits and giggles.
Asgore may be willing to hurt children, but he clearly hates doing it. When he faces the main character, he refuses to look them in the eyes out of shame.
Examining the Monster Kid on a No Mercy Run will show the message "Looks like free EXP". Even though he is saved by Undyne Taking the Bullet, you must attempt to attack him to complete the run.
In One Chance, if you spend the last day where everyone is alive and well at home then helping to create a cure, a scientist will try to murder you as all the other had already died. If you block the attack, he will run away. Returning home however, you find the bodies of your wife and kid, along with the coworkers' corpse.
The enemies of INSIDE have no qualms with siccing attack dogs or firing guns at the protagonist, who is at most ten years-old.
In Tyranny, Kyros' laws make this illegal; the young are to be protected. At least, if they're citizens of Kyros' empire; non-citizens have no rights. In the Blade Grave, on the other hand, ending the Edict of Storms, which will otherwise continue to devastate the land and kill thousands, requires the eradication of the entire Regent bloodline. Including an infant still in her crib. Forcing the Fatebinder to suffocate the child or have a companion end their life... unless they can find an alternative.