When talking with Salvatore, Brother William seems surprised to find that Salvatore eats the rats he catches. Rats were commonly eaten by the poor in the Middle Ages. Brother William would have been more surprised had someone catching rats not eaten them.
The secret message on the parchment is exposed three times. The translator heated it to reveal the location of the library, William of Baskerville heated it again when he was in the scriptorium and yet again to show the others the message. When a message is written in lemon juice, heating it will cause it to become exposed because the sugar in the juice is caramelized and thus would not disappear again.
At several points in the film, individuals enter another's cell (room) rather freely. In ancient religious rules, entering another monk's cell without permission was normally forbidden, even punishable by excommunication.
Near the beginning, when the abbot comes into the cell where William of Baskerville is alone (Adso having left to seek the necessarium), the abbot greets him with "Pax vobiscum" - but this is the plural form for addressing more than one person. It is taken from the start of each Holy Office, when the abbot would be addressing the choir monks as a group. He should have said "Pax tecum", with William's response being correct as "Et cum spiritu tuo".
The abbot greets Brother Cuthbert of Winchester as "Your Grace". This form of address would only be correct for a high level noble (who would not be a member of an order of poor monks) or in England for a high member of the Catholic Church. It would not be appropriate for a monk in Italy.
(at 1 hour and 55 seconds) When the body of brother Berengar (who was found drowned in the bath) is lying on a table for examination, a shaft of light creeps over the actor's eye, causing him to blink. This occurs immediately after the close up view of his stained left hand.
At 1 hour, 48 minutes and 13 seconds, when brother Jorge de Burgos is speaking in the chapel, the "preservation, I say, not search for" shot shows his right eye a clear. The contact lens to make his eye like a blind man is missing. It is back with the next closeup shot of his face and missing again at the next shot showing his face when he speaks "that the bloody-eyed and cloven-hoofed Antichrist".
The goof item below may give away important plot points.
The first time in the library, Adso unravels his robe to help find the way out. Although Brother William and Adso are shown with only one robe each, Adso's robe is back to normal after exiting the library.