* [[spoiler:Hex]]'s HeroicSacrifice in Season 4.
** [[spoiler: "Bob, I have always loved you. Dot, take care of him, and be happy..."]] *sniffle*
* The ending of "Web World Wars": [[spoiler:Bob is shot into the web as Dot stares without being able to do anything other than scream Bob's name, then proceeds to close the portal. Heartbroken, Dot looks down, notices Bob's keytool that had Megabyte had just crushed and tossed aside on the floor, picks it up and painfully looks at it. Later, when queried about what happened to Bob, Dot simply says "I'm sorry" and places the broken Glitch on Enzo's hand, prompting the young sprite to scream a BigNo before breaking into tears.]]
* Two moments from "Game Over":
** Megabyte orders his fleet to attack the principal office. The fleet is cut off and destroyed, yet the viral binomes go down fighting, the commander shouting his loyalty to Megabyte. Megabyte, huge JerkAss that he is, is unaffected, but [[WhatASenselessWasteOfHumanLife Dot weeps over the slaughter]].
** The ending. Made even worse by everybody's shocked silence (19 seconds, of everyone in the War Room just shocked and staring at Dot in disbelief and even mild incomprehension) after they hear the words [[spoiler:"GAME OVER. USER WINS."]], only to have it broken by Dot's [[BigNo grief-stricken scream]].
** Surprisingly, Slash gets a minor moment when he laments the way things have been going in Mainframe lately.
--->"Bob always stopped us before we could do anything ''really'' bad. Now, nobody does."
* "Megaframe." The moment where Enzo, now Matrix, is reunited with Dot, usually starts the waterworks up pretty quick.
-->'''Matrix:''' Uh, hi..... sis.
-->'''Dot:''' ...Enzo? ''[bursts into tears] ...Enzo! Oh, '''Enzo!!!''' [[[CryIntoChest hugs him]]]'' I thought you were nullified! I thought I'd lost ''everything!''
** One thing about Matrix and Dot's reunion that always seems saddening is the short look of confusion on Dot's face. Either she doesn't recognize her brother at first, or she's in denial that it's really him and it HURTS to see. Even worse, Matrix grew up at an accelerated speed so he's not supposed to be anywhere near as old - something that almost certainly hit Dot.
** The tracking shot of Mainframe in ruins, with Matrix, [=AndrAIa,=] Bob, Captain Capacitor, and Frisket just staring in horror. Worse when watching immediately after the preceding episode's triumphant ending of our heroes finally managing to make it home.
* Hex curled up in the Fetal position and shrinking away from Scuzzy at the end of "Painted Windows".
* Hex's reaction to her masks being fused together, effectively bringing her full sanity for the first time in her life.
-->'''Bob:''' How do you feel?
-->'''Hex:''' For the first time... WHOLE.
* Though it leads to a HappyEnding, in ''End Prog'' the forced system crash Bob implements by allowing the User to win a Game by keeping everyone out of it. The reactions from the Sprites and Biomes as their world is ending all around is devastating, especially an image of a Little Girl Biome, all alone without her parents or anyone, cradling her doll as the System Crash is announced.
* While Enzo and [=AndrAIa=] are together at the end of her debut episode, that [=AndrAIa=] is a copy. The original [=AndrAIa=] left forever with the game cube and is all alone.
-->'''[=AndrAIa=]: '''Goodbye, my friend.
* Frisket drained of energy by Gigabyte. Sure, Bob immediately reassures Enzo and [=AndrAIa=] that he'll be fine, but it doesn't make the horror of seeing their faithful companion downed in an effort to protect them.
* Enzo looking around for [=AndrAIa=] in "Between A Raccoon And A Hard Place", after it looked like the game had taken her with it. It's a brief moment, but the look of despair on his face and the tone of his voice as he calls "[=AndrAIa=]!" really help convey the desperation the young sprite was feeling.