At the end, there's an extended amount of time where it appears that Shrek failed. And, technically speaking, he was dead for a while. This movie being Darker and Edgier than the others, it wasn't unbelievable that they'd do it.
When the alternate universe starts falling apart, even knowing things are going to be set to rights, Fiona calling and reaching for Puss when he dissolves strikes a chord for many pet owners.
There's also the moment somewhere right after he's beginning to understand what his impulsive wish did - he finds his daughter's doll still in his back pocket from where he'd put it very early on in the film, and the look on his face as he realizes that she doesn't exist anymore is absolutely heartbreaking.
The song "Fiona Doesn't Love Me" and the accompanying scene. Shrek thinks all he has to do to break the spell is get Fiona to kiss him, par the rules of True Love's Kiss. He manages this, but because she doesn't know him that well and thus doesn't love him, nothing happens. Donkey asks him what went wrong, and he has the most despairing look on his face as he says, "But Fiona doesn't love me."
Alternate!Donkey's fear of Shrek is sort of a Fridge Tear Jerker. The Donkey that Shrek knows was afraid of him for maybe five seconds when they first met, then decided they were best friends and couldn't be scared away after that. Considering this misadventure was set off by Shrek missing the days he was feared, Donkey running away from him in terror even after Shrek tries to explain and win his trust by singing must be a nasty hit to the irony bone.