Smoke and mirrors. Optical illusions. Puzzles. Riddles. Trap doors. Hidden sharp objects. Hidden blunt objects. Needing multiple people to complete something or turning multiple keys or keylike objects from other parts of the story perhaps?
The room is filled with a deadly gas. Either it is built so that it vents into the room naturally from deep under ground, or a diffuser is periodically refilled by whoever controls access to the room.
The McGuffin is the antidote to the gas, which you must obtain and administer before entering.
One way of getting a very localised danger like this would be to put it near radioactive ore deposits? You can only go in with a lead shield, and have to angle it correctly
The room is built on the inside of a giant snail's shell, if you give it a special kind of lettuce the snail will retreat and clear access to the room.
Simply put: it stinks, like bad. The room is hidden under the largest city in the land, only accessible by spelunking through vast distances of underground sewers that smell like decomposing waste. This smell-field ensures only the bravest and most dedicated adventurers ever travel to the room, and none who attempt it enjoy talking about it.
Is it on display in the bottom drawer of a locked filing cabinet stuck in a disused lavatory with a sign on the door saying, "Beware of the leopard"?
More seriously, just make it an incredibly uninteresting door that no one even thinks about, which everyone thinks is like for plumbing or linens or something boring and irrelevant. Then paint the door in whatever Disney NoSeeUm color is most appropriate.
Some old palaces in Japan have "nightingale" floors that purposely squeak to keep people from sneaking in. Could be a security feature outside your room
Boobytraps. Rumours about a curse/ghosts/entry fee. Pretend there's something boring in there. Allow entry, but make the process the full force of German bureaucracy (you need to file form A on a blood moon, to get form A, you need document B2, no not that one the green one, you can get it from Location C but that's only open once a month for half an hour, and if you don't wear pink they won't help you).
You can spread powder on the floor so you could see if someone had stepped in. It wouldn't keep them out unless they didn't want anyone to know they'd been in there.
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