What makes a film disturbing is when you feel you are in the hands of an irresponsible filmmaker - one who takes a horrific situation (which most of us aficionados consider fun escapism) and pushes the pain/fear/suffering into extremely realistic territory.

And long, subjective takes, not quick loud edits, bring that sense of verite to life.

totally. unknown actors help a ton.

I think showing anything that makes people realize how depraved and disgusting their fellow man can be does the trick.

Atmosphere and mood are one of the biggest parts. The way high tension was filmed, the atmosphere was awesome. I was sitting on the edge of my chair. The music was perfect as well.
But also to be disturbing there has to be the element of reality. Thinking this could happen is the bit that disturbs you. This is what made the August Undergrounds and Irrevrsable disturbing.
Freddy v's Jason however doesnt bring this ;)

Killing children and cute animals are always big ones. I liked the way a kid was used in the original Assault on Preceinct 13 and animals in Equilibrium.

Yeah but it really depends on what it's used for. In AoP13 it was used to create the story, to give the father a need for revenge so the gang could be incited to hunt him down. If it's a simple slasher flick and they show a kid get killed in a more dramatic way (the big eyes, the sappy music) than an adult then yeah, it's just the lack of good ideas. I think the problem with killing children (in movies) is that they treat it differently than the adults. If they showed true equality in the brutal slaughter of adults AND kids I think it would be more disturbing and less cheap emotional manipulation.

That's why it has such an impact, it's the closest thing they can show comparable to killing an angel.

Yep, just using something proven to draw from the audience the emotion you want instead of trying to develop something new and powerful.

I think that it needs to have a sense of reality, which is why Last House On The Left was disturbing...

unexpected twists