The ending of "Web World Wars", after Bob is shot into the web.
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 Jerk Ass that he is, is unaffected, but 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 each other in disbelief and even mild incomprehension) after they hear the words "GAME OVER. THE USER WINS.", only to have it broken by Dot's 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."
The moment where Enzo, now Matrix, is reunited with Dot is a tear-jerker of a heartwarming sort.
"Uh, hi..... sis."
One thing about Matrix and Dot's reunion that always gets this troper 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.
It doesn't help that Matrix grew up at an accelerated speed so he's not supposed to be anywhere near as old - something that almost certainly hit Dot.
Hex curled up in the Fetal position and shrinking away from Scuzzy at the end of Painted Windows.