In New Moon, British actor Robert Pattinson returns as the blood-sucking vampire Edward Cullence in love with the beautiful Bella Swan.
The movie has already broken box office records as the highest single-day earner after pulling in $62.2 million on its opening day.
Monsignor Franco Perazzelo, of the pontifical council of culture, said on Friday that the theme of vampires in Twilight "combines a mixture of excesses that, as ever, is aimed at young people and gives a heavy esoteric element".
"This film is nothing more than a moral vacuum with a deviant message and as such, is something, that should be of concern," he said.
Last month, the Vatican warned parents that Halloween was had an "undercurrent of occultism" and was "absolutely anti-Christian".
The Vatican advised parents instead to "direct the meaning of the feast towards wholesomeness and beauty rather than terror, fear and death".