The Artifacts in the Library are: Tesla's Death Ray, The Holy Grail, Little Boy (atomic bomb), The Ark of the Covenant, The Golden Fleece, King Midas, Excalibur, the Goose that laid the golden egg, Pandora's Box, the real Mona Lisa (a copy is in the Louvre), a Unicorn, the Rocketeer's Jetpack, Ali Baba's Flying Carpet, and the Spear of Longinus (Spear of Destiny).
When Flynn and Nicole are walking through the Amazon Jungle, Flynn says something to Nicole, to which she replies "Be still my heart". ER: Be Still My Heart (2000) was the ER episode (season 6 episode 13) where the character played by Noah Wyle (who plays Flynn in the Librarian) is stabbed and almost dies.
In the jungle when Flynn reads Nicole's (played by Sonya Walger) personality, he says among other things that her father is Argentinian but that she never learned Spanish. Sonya Walger's Father is actually Argentinian however contrary to the film she is fluent in Spanish.
The two lions seen on either side of the steps down from the elevator to the secret library are not duplicates of the two lions, popularly known as Patience and Fortitude, outside the New York Public Library. Rather they are copies of the lions on the monument to Pope Clement VII in St. Peter's Basilica, Rome. They were sculpted by Antonio Canova between 1783 and 1792.
Even though they did not share any scenes, Bob Newhart and Noah Wyle shared one previous project before making this movie when Newhart guest starred on ER (1994) (co-starring Wyle) in 2003 as an artist going blind and contemplating suicide, a rare dramatic role for the legendary comedian.