It feels a little dull. None of your colour ramps seem to employ hue-shifting, that is, making the highlights and shadows different hues in addition to giving them different values. Adding hue-shifting can bring a lot of life into your work, I recommend adding some! For example, you could make all the highlights a little yellower/warmer (since the sun is lighting them) and all the shadows a little cooler/bluer, since they're getting the ambient light from the sky.
On a related note, pure greys tend to look rather boring. Clouds, especially on sunny days, are usually not grey, they usually have blue shadows (because the ambient light diffused by the sky fills in the shadows).

Some of the trees don't contrast well from the sky, because they're nearly the same value. Try using a lighter sky and some darker, less saturated greens for the trees. The narrow green trees also look neon and almost radioactive because they're so saturated and because they're lighter than the sky in places, I think slightly less saturation and darker values would really help.

The interior of the bedrock(?), which is presumably the least important thing to look at, is currently hogging all the attention because it has large, clear shapes with relatively high contrast compared to everything else. I think a solid colour with just occasional touches of detail would help keep the focus on the surface and in the dirt with the roots, which I'm guessing is where the player is meant to look. Also, if it is bedrock, it shouldn't be a loose pile of rocks, but rather something solid and possibly layered; the loose piles only happen where the bedrock is exposed to weathering, not where it's covered up.

The gradients on the trees (leaves and trunks) and clouds feel very "programmer art"-ey, and don't match the more interesting textures you're using elsewhere. I think those could really use some additional work. Even if they're procedurally generated, you could use more interesting base sprites/shapes as building blocks, they don't have to be solid/gradient shapes.

Here's a quick edit (mostly using some blending layers rather than manually-editing colours, I'm lazy and your palette is very large xP) that makes the dark colours bluer, the light colours yellower, and desaturates the neon greens:

I also replaced the grey rocks with some dark bedrock, as I think it creates a nicer visual hierarchy, letting the important stuff (the dirt and the stuff directly above it) be framed between two relatively flat expanses of colour (the sky and the bedrock). I doodled in some slanting strata freehand, but the same effect can be achieved with tiles like you're using, and can look much nicer and more rock-like than my doodles.

Please don't use the "width" parameter when posting images, just use plain [img]. The fixed width prevents zooming, which makes it harder to look at your image and give you more detailed feedback. The "img width" feature is meant for clickable banners and such, not for sharing art for critique.

MysteryMeat is right, avoid having highlights everywhere. Use them to draw the viewer's attention to important things, such as the parts of the environment that the player can interact with directly (e.g. the tops of platforms).

The edges of your tiles are perfectly smooth, which makes all your hard work on the textures in the interiors of the tiles feel a bit wasted. The edges should be as textured as the interior, since they're the same material.

The edge tiles are distinct from the interior tiles, which means they're an opportunity to create variation and a more natural look. By copy+pasting substantial portions of different tiles, you're squandering that opportunity and making things look more copy+pasted than they need to. That's fine for tilesets that have cartoony textures or that are meant to depict repeating textures, but you seem to be going for something more natural-looking, something that should have its repetition minimised.

The diagonal boards have banding, that probably makes the jaggedness more apparent. Reduce the banding and they'll probably look better. 2:1 and 1:2 slope steps usually look fine without banding, but if you feel the contrast it too high even without banding, perhaps adding AA could help. Also, remember you can add some dips and "overlapping" dirt on them, hiding the jagginess.
To get rid of the banding with minimal work, you could make the sleepers (including the horizontal and vertical ones) mostly be that shadow colour, with smaller/fewer highlights. That would also help the sleepers look like they're in the ground rather than lying on top of it.

Your terrain isn't really 1:1, the rock shapes on the ground being wider than they are tall suggest a lower camera, something closer to 2:1. The fact that we can see so much of the vertical walls without them being squished vertically also suggests a lower camera. The character movement might be 1:1 (but that could be changed if you wanted to), but it's not likely to be noticeable as a problem.
You're right that most players won't think about it. Sword of Mana mixes 2:1 and 1:1 and even lower, almost side-view cameras and no one bats an eye xP

I think a big part of the problem is the outlines. Outlines look like shadows. If something's meant to be flush with the ground, it wouldn't be casting a visible shadow underneath itself. Removing the outlines around the sleepers helps a lot, it makes them look buried in the ground and almost flush with it. The rails can keep their outlines, since they're lying on top of the sleepers and are elevated slightly above the ground.

Here's a pair of edits.
The top edit removes the outlines from the sleepers, and makes the sleepers a little darker (fewer highlights), which makes them look a little dirtier and lower in the ground.
The bottom edit adds some piles of dirt around the rails (achieved simply by erasing parts of the rails, so the tiles can work on any terrain). Note the lack of outlines around these piles - the dirt is flush with the wood, so there's no separating shadow.


If you want to get very specific with the tiles, you could adjust the colour of the wood to be a little closer to the colour of the ground, to make it look like the dust has permeated or covered the wood.

The mouse rounding might be a screen scaling issue. Aseprite has separate screen scaling and UI scaling, make sure screen scaling is at 100% (and set to UI scaling to 200% or whatever you like). The default settings have it backwards for some reason.

I've seen a few people who dislike Aseprite's pixel art UI, so you're definitely not alone in that. I'm no fan either, but it's not a bother to me since it's just a visual skin on an otherwise convenient UI (at least with 100% screen scaling xP).

UI and workflow count for a lot, so I don't think it's petty or stubborn to want to make your own thing if using/extending existing software isn't a good option.