Cassidy sacrificing herself at the end of "The Midnight Zone".
Mr. E's flashback to him and Pericles meeting when he was a kid in "The Gathering Gloom". Especially his line before it: "Were we ever once so young?" Made worse when they reunite as friends at the end of the episode...while plotting Cassidy's murder. How they have changed.
Mr. E in general is a tragedy. His relationship with Cassidy crumbled, but they never stopped loving each other. Once he graduated high school, his Big Eater tendencies caught up with him and transformed him from a skinny, goofy, Shaggy-esque nerd into a grotesquely obese, grouchy man who still carries severe emotional scars for the rest of his life, his once-promising life at the end of high school completely destroyed.
Fred's reaction to finding out his entire life was a lie in "All Fear the Freak". Welker's voice acting really sells it.
The entire ending to that episode qualifies.
After Fred unmasks him, Mayor Jones can't bear to make eye contact with anyone, particularly not with Fred, without shame.
Remembering that earlier on in the episode, he seemed sad while looking at that photo of Fred's supposed mother. In hindsight, he was probably sad because he was scared he would lose his son if he discovered the truth about him and what he did. At the end of the episode, despite Fred being visibly angry and hurt by his confession, Mayor Jones still believed he would stay with him. In fact, their last conversation of the season is this (as well as Mayor Jones's last words this season as well as for a while):
Fred: So all of this for a treasure no one's even sure exists?! Where'd you put the disk, in your pocket? [Fred discovers Jones's pocket is empty] Mayor Jones: It's gone?! We have to find it! That pieceis priceless![Fred pushes him to the ground and runs away] Fred! Fred!
Hearing Sheriff Stone cry and say to Mayor Jones, "Tell me this isn't true!" at the end.
There's something both tearjerking and yet heartwarming in how Fred knewthat he could get his false dad to help with the plan, while his real parents would try to steal Mystery Inc.'s disk pieces instead in "Wrath of the Krampus"
Also both is Fred, who has his real parents, admits that while his fake dad was harsh on him (and not his real dad), he still got attention from the man whereas Brad and Judy mostly ignore him unless they're explicitly using him, to the point where poor Freddie actually feels a void.
It's even more sad when you realize that at the time, Brad and Judy actually did love their son, but since they didn't raise him, they never learned how to actually be parents. Aside from bonding with him over traps, the only parental thing they tried to do was to protect their son from the treasure's curse, which only ends up leading them down a path of corruption by the cursed treasure instead!
Scooby crying for Shaggy after Shaggy gets kidnapped by the Weird Gnome in "Grasp of the Gnome".
The whole story behind Dr. Zin and his daughter in "Heart of Evil".
The Horrible Herd. There's a scene that seems to be a direct shout out to the most traumatizing moment in The Lion King. Except Mufasa's been replaced by Nova. Scooby is understandably devastated. She's found alive, and the Gang rush her to the hospital, but considering how badly she's injured, one wonders if Friar Seras was talking about Scooby when he said "the dog dies"...
The next episode, The Dance of the Undead, features Nova in the hospital. Scooby visits her no less than five times a day, according to Shaggy. In the end, she flatlines.
The first few minutes of The Devouring give us a quick one, but a gutpunch still. Ricky stares thoughtfully at photos of Cassidy before confronting Pericles about going too far. It's starting to look like the exploitation of his company isn't his only regret. The fact that Cassidy had been willing to work for him after all those years and despite her misgivings was a big hint that their past affection hadn't completely died out on her end, and maybe this was one from his. Too bad it came too late; with her death confirmed on Twitter, he'll never see her again. Unless Pericles gets a little too eager with the cobra venom...
In "Nightmare in Red" we learn the spirits of El Aguirre and his men don't want the Nibiru entity to be destroyed which would lead to them being freed because they believe they don't deserve salvation after the things they did. Aguirre makes vivid, horrified recollections of the carnage and he and his men inflicted, while the background music shifts to the sounds of swords clashing and people screaming. This really drives in what a heartless, vindictive monster the creature inside the sarcophagus must be if it is capable of corrupting so many individuals to that extent. Aguirre and his men have spent centuries in limbo believing themselves to be complete monsters because of what the entity did to them.
Also the fact that the Gang believes they are probably only together because of the being, even going as far as to believe they probably would not like each other if it wasn't for that thing manipulating them into being a group.
In Gates of Gloom, the Tearjerker entry for The Devouring gets so much worse. What we thought was Ricky mourning Cassidy was actually him wondering why he hadn't heard from her. Ricky's been unaware that his actions led to his ex-girlfriend's death this whole time, and has been obliviously cooperating with her killer. Upon finding this out, he steadfastly refuses to obey Pericles's order to allow the massacre of every resident of Crystal Cove...until Pericles tortures him with snake venom, forcing him to comply.
In the next episode, before she dies, Hot Dog Water tells him she thought he'd be better than this, to which Ricky replies "So did I." Afterwards, there's a scene where Ricky says that none of them will make it out of this alive, then he laughs and says he's so happy about that fact. Ricky has become so disgusted with himself and how far he's fallen that's become a Death Seeker.
On a lesser note, Freddy sacrificing the Mystery Machine. Seeing the iconic vehicle of the franchise blow up like that...
Hot Dog Water's death in "Through the Curtain", with the camera cutting away to the gang right before the gun fires. Velma's reaction to the sound of the gunfire in the distance and seeing Scooby turn and whimper adds to the punch. "Just keep going, Scooby..."
From "Theater of Doom": "The dog dies? Is he talking about me?"
What about the Entity devouring almost everyone the gang's come to know and love in "Come Undone"? It was bad enough at first that we saw helpless incidentals getting snatched from the streets & their own homes before getting eaten, and then we see Entity grab someone we do know—Mayor Jones (with Fred reacting in shock, especially because they had just reconciled), then the Sheriff, and then Mayor Nettles, with the Mystery Inc. right behind her as she gets grabbed.
When it's all over, it's kind of sad that all of Daphne's sisters are stripped of their careers and intelligence, which seems to have come from the negativity in the town. Delilah, especially, because she at least seemed nice, if not a tad gruff.
In general, seeing how the curse affected everybody around the gang and negatively influenced so many people's lives is pretty bad.
To elaborate, this is the main reason the gang keeps facing people with supernatural gimmicks. The Evil Entity would influence their minds and drive them to villainy in order to keep Mystery Inc. active. Meaning that almost everyone the gang faced (including from the previous series) were probably normal people whose lives were ruined to keep the Entity's plan in motion.
And don't forget at the end of the series, before receiving a disc from the new Mr. E, the Mystery Inc gang feel like they're outcasts because not only did they see everyone and everything dead in their old universe, but because of what happened after defeating Nibiru, none of them feel like they belong to this world, especially because they feel that the unaffected love ones they saw were not the same ones they actually cared about. You can't help but give these five a hug.
Going by the show being a Stealth Prequel to Where Are You?, it suggests Fred legally changed his last name to Jones, because he was too used to being Fred Jones, Jr., son of Fred Jones, Sr.. He never got to live the life of being Fred Chiles, son of Brad Chiles and Judy Reeves, even if Ripple Effect-Proof Memory comes into play.
In a meta-tearjerker, hearing Casey Kasem as Shaggy's father one final time in the series finale with the knowledge that the episode will be his final involvement in the Scooby-Doo franchise ever since the franchise started nearly 45 years ago. Kasem passed away about a year and a half after the finale aired, but his final words in the franchise are his character telling Shaggy how proud he is.
After the mystery in "Dead Justice" is solved, Fred goes looking to his dad for a So Proud of You moment and is practically begging for it. Instead, Mayor Jones just brushes him off and walks away.
And then followed up two episodes later when it turns out that Mayor Jones is Fred's kidnapper, having abducted him from his real parents as a hostage, all while ripping apart the original Mystery Inc. As far as Fred knew at the time, he could've grown up with love and approval he's yearned years for, but his supposed father figure was always a Jerk with a Heart of Jerk—ignoring that what confirmed Fred's suspicions that Mayor Jones and the Freak were the same person was when he saved his false son from dying just minutes earlier.
It isn't until nearly a complete season later that we learn that Daphne was right: Mayor Jones really did love Fred. But the curse made him inept at doing so due to sealing away his good subconscious, yet this reveal also implies that his good side managed to make itself prominent when Fred nearly fell to his death back in the Season 1 finale.
Danny Darrow's life. When he was a child, he and his family went crazy with greed after finding a piece of the planespheric disc, and their house sank underground where he was forced to live out his life (about 75 years), obsessed over the piece, while he and his family grew old, and they all died, leaving him alone. In one scene, the gang sets a trap, and Scooby lures him in while pretending to be his mother, he responds with a genuine hopeful smile and says "Mommy, you came back to me?" Even though she died years ago.