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The only reason the Chaos Marines haven't smashed through the Eye of Terror long ago is because at the mere mention of "Black Crusade" the Imperium devotes a metric gakload of forces to blunt it, and have redirected forces that originally were meant to combat other threats like Tyranids towards the Eye of Terror to ensure the Marines don't get a foothold.

And that's just the Marines.

Chaos is the biggest threat because they are very active at the moment and beyond that humanity has no way to defeat them.

I'd say the largest strains on the Imperium are Chaos and the Orks.

Chaos is a threat because it's right in the Imperium's bonnet, and it has united numbers. The Necrons aren't particularly numerous, while the Tyranids are diverted into attacking other alien species (Including the Orks and Tau). Chaos is also capable of contagious diffusion; as soon as they get out of the bottleneck, many people will start switching to Chaos worship as news of the Dark Gods' success spreads.

The Orks just live everywhere. They might not usually create wide-ranging campaigns, but they don't need to. The sheer amount of Ork worlds means that there's a very likely chance you're living next to one. And it doesn't take some huge Ork military force to secure a planet that's unprepared.

In the short term, probably Chaos due to propensity for aggression and proximity to major Imperial territories (Eye of Terror, etc). But in the long term, It's got to either be the Necrons (depending on how many more there are still dormant) or the Tyranids (most recent codex suggests that the current hive fleets are a vanguard for the real swarm).

That said, the Orks are sort of the standard galactic cockroach that you just can't seem to keep down for long.

chaos is the biggest threat

I'm gonna say Necrons because at full power the only thing that can defeat a Ctan is the Talismans of Vaul, which the Imperium has no idea how to use.

If the orks ever managed to get together, they would crush
the u'mies, but right now the largest threat is from chaos