The title is a pun on that of the film noir "Guest in the House" (1944).
The victim-guest's assigned room number, 666, is a sly reference to "the number of the Beast", i.e. of the Antichrist, in the New Testament Book of Revelation. This doesn't bode well for the room's occupant.
The name of the "Gland Hotel" (0:37) is a rather peculiar pun on that of the title establishment in the movie drama "Grand Hotel" (1932).
As per his contract, Mel Blanc is the only credited voice actor in this cartoon. Yet, of the three characters appearing in the short, only one (Daffy) is voiced by Blanc. Both Elmer Fudd and the hotel guest are played by Arthur Q. Bryan.
Despite naming all of the keys on his ring, Daffy does not actually unlock the guest room door onscreen.