Over its 14 seasons, Supernatural presented us with many characters, amd it can be hard to determine the main characters aside from the brothers, Sam and Dean, Cas, and Jack. Still, there were characters that were closer to the story in recent seasons, and others that had a lasting effect on Sam and Dean, making them main characters.
That said, all of these characters have performed good deeds (yes, even Ruby and Lucifer), and all of these characters have done horrible things (yes, even and especially, Dean and Sam). Even the angels are tainted in Supernatural.
Lucifer: Stealing His Son's Power
We would expect everything that Lucifer did to be bad, so it is hard to choose the worst. Could it be when he tortured Sam? Could it be when he killed Cas? When he brutally killed Rowena? Or when he treated Crowley, literally like a dog? Then, there were all the angels he eviscerated. All these and more are horrible, but the worst thing he ever did was stealing Jack's (his son) grace. It had seemed that he was interested in his son, but his son was just another way to get power.
Lucifer's Human Vessel, Nick: Dooming His Wife
When Lucifer leaves Nick's body, Nick comes back. First, he begins to search for a way to avenge his family, his wife, and child who had been murdered. He kills people and demons to do this, sometimes innocent people, but this isn't his worst thing.
Finally, he goes back into his old house, and there he sees his wife's ghost. She tells him that she's trapped in the house due to the unfinished business of her murder and his path with Lucifer. She begs him, "Reject Lucifer right now. If you do, I can leave. I can find peace." Nick won't do it, turning his back on his wife.
John Winchester: Getting Jo's Dad Killed
Normally, we would say that John's worst thing was how he raised his boys, but his teachings made them into men who can save the world. If he hadn't given them the tough love and the field experience with hunts, they might very well have turned out like their half-brother, Adam, first killed by a standard ghoul.
On top of hist list of worsts is getting Jo's dad killed. From what Ellen, Jo's mother, says, her husband went on a hunt with John, and John used him as bait and left him. Ultimately, this left Jo without a father, and Ellen without a husband.
Rowena: Attack Dog Spell
When we are introduced to Rowena in the series, we see a quirky red-haired witch intent on creating her own coven. While she's powerful, the Grand Coven rejected Rowena for her odd and often extreme ways. At first, she seems decent, rescuing two prostitutes and wanting to introduce them to power. However, when threatened, she uses one as a prop for her escape. She turns the woman into an attack dog, who quickly bleeds out after the spell runs its course.
Crowley: Killing Those Sam and Dean Had Saved Before
Since we like Crowley in the later seasons and consider him more of an ally to Sam and Dean, we forget that he started out as a big bad. In Season 8, he threatens to kill someone they had previously saved until they stop the trials. One of his victims is Sarah Blake, a charismatic side character we met and loved in Season 1. Had Sam met her at a different time (he was still grieving Jess), they might have become an item. Horrified, Sam has to watch Sarah killed despite all the wards and protections he and Dean put up to try to protect her from Crowley.
Chuck/God: Bringing The Dark Spirits
Chuck/God has been mainly a side character even though he is the mastermind behind it all. In Season 14, he tries to manipulate Sam and Dean into killing Jack. Sam notices how Chuck watches with glee as Dean almost kills Jack. In the end, Chuck kills Jack. Sam and Dean discover that Chuck has been playing their lives for entertainment all this time. Angry with the brothers, Chuck decides to bring back all the dark spirits they vanquished over the seasons, even back as far as Season 1. He's decided to end this world and to make that ending painful and tragic.
Ruby: Using Sam to Bring Lucifer
Ruby seems the anomaly, a demon with a heart of gold. She trains Sam to take down Lilith, feeding him demon blood to make him more powerful.
In the end, she wanted Sam to kill Lilith because she knew that Lilith was the last seal needed to be broken in order to bring Lucifer into this world. She played all sides convincingly, betraying both brothers in the end, who had come to trust her.
Bobby: Becoming a Vengeful Spirit
After Bobby dies, his spirit stays attached to his whiskey flask, which Dean keeps in his honor. Initially a helpful entity, Bobby points out research that helps the brothers in their demon hunting. However, he starts to develop into a vengeful spirit, like the ones that they all have vanquished before. Right now, he is still Bobby, but Sam and Dean realize that he won't be for long if he doesn't cross over.
Mary Winchester: Killing American Hunters
The British Men of Letters recognize Mary as the perfect tool for them to get rid of the American Hunters. Not only is Mary a good and strong hunter, but she is also trusted by the other hunters. This means that when she shows up at the doorstep, they will let her in. While Mary was brainwashed to do this, she still did it, and she effectively whipped out many hunters and allies.
Jack: Killing Mary Winchester
While Dean feared Jack from the start since he was the son of Lucifer, Jack initially is good. When he has his powers, he saves people constantly and sees Dean, Sam, and Cas as his idols. Even when his powers are taken away and he becomes fully human, he still puts his life on the line to help others.
But a soulless Jack is different. It's not that he is bad, it's that he doesn't feel morality. He kills Mary because he wants her to stop talking. He didn't intend to kill her, and he even tries to bring her back. However, he can't, and Mary remains dead.
Cas: Tries to Become God
Season 7 Cas takes in souls, making him more powerful. He becomes full of ego and power. This leads him to kill the angels and people that don't support him. He becomes dangerous in his own idea of right and wrong, believing that he must rule with a firm fist. Later, he does sacrifice himself (luckily, he survives), but for much of Season 7, he is the villain.
Dean: Kills Amy Pond in Front of Her Son
Early Dean had a strong sense of right and wrong, seeing the world only in black and white. When he finds out that Amy (a kitsune) was killing to help her sick son (another kitsune), he sees her as evil and without much hesitation, kills her. She had been a friend of Sam's when they were teens, and she saved Sam. She lived her life trying to live off dead blood, until her son got sick. Sam tells Dean to let her go, as she stopped killing and won't again. Dean goes anyway and kills her in front of her young son.
Sam: Kills Dean's Daughter and Feels No Remorse
Dean has a one night stand with an Amazon woman (though he doesn't know that she is an Amazon at the time). This results in her conceiving a child, who grows to be a 16-year old girl in only a few days. As part of her initiation, she must kill her father. Sam stops her by killing her. While this is bad, what is worse is that he tells Dean that Dean went soft by not doing it himself.
Adding one more thing to our favorite brothers, they also left Adam, their half-brother, behind.