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
Depends on the context, like what world it is and where the room is located, and so on.
I suppose it's in a building that people have access to except for that one room, and that it's a world where, while not particularly sophisticated, explosives exist, so it should at least have very strong walls. If magic exists you might want to avoid using it for this purpose as it often becomes a "because" kind of explaination, but you can use it as a tool (e.g. an obstacle that can only be overcome with powerful magic, and the magicians who are capable of such power can be counted on one hand).
If magic is a no-go, maybe it simply requires a specific kind of key to open (but maybe it's too high tech for your world?)
Key?! What kind of futuristic sci-fi do you think I'm writing?!
But seriously speaking, I think I'll make it so that you have to visit a cave with a stream flowing through it, grab a fish from the stream, maybe do some rite and then the fish burps a key out.
Set up a monastery in the surrounding building whose religion is centered on only letting in the one person. The 'key' is a complex ritual (like happens nightly at the tower of london) with lots of phrases and movements to learn. The ceremony happens somewhere dangerous to the person requesting access.
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 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.
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).
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
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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