This is based on opinion. Please don't list it on a work's trope example list.
Tearjerker / Spiders And Magic Rise Of Spider Mane
Anything concerning Peter's past and how he sees himself as nothing but a failure because of it, is heartbreaking, but its even more so from the point of view of his new empathetic friends.
Adding to the above, the chapters where the Equestrians get to see all of Spidey's memories is particularly difficult—not because of all the failures, but because we see his greatest victories as well. Not just the usual, like overcoming the black suit and later Venom himself, but also one of his biggest, if not THE biggest victory in his career—holding the Infinity Gauntlet and all its world changing power, and letting it go. He saved all reality, but because of two mistakes—only one of which could be considered even remotely his fault by any sane definition—he thinks of himself as a failure and a horrible person. Peter's a horribly broken person, really.
Applejack's backstory in chapter 39.
The cemetery scene in Chapter 46.
Peter's death and Twilight's reaction to it, in chapter 54.