The filming of the final "Ave Maria" sequence was plagued by mishaps. To achieve the effect of moving through the scene, several panes of painted glass were used. The whole setup was over 200 feet long, and had to be redone three times. The first time the wrong lens was placed on the camera, and the subsequent film showed not only the artwork but the workers scurrying around it. The second time around, an earthquake struck the studio, and the shot was once again scrapped. The next morning, the shot was redone, the film was shipped to the lab, processed, and couriered to the premiere in New York where it was spliced into the final print with only four hours to spare.

The first major Hollywood film to be released with no written credits appearing onscreen except the intermission title card.

In the "Pastoral Symphony" segment there was originally a scene showing stereotyped Black assistant centaurs shining the hooves of white centaurs. The chief of these was Sunflower, who had a very stereotypical look: big, red lips and wild messy hair. It was not until the 1969 re-release that this was thought to be objectionable, and all subsequent releases until 1980 had an abrupt cut at this point. Every subsequent release after 1990 includes the scene, but with the section blown up so that it only shows the faces of the white female centaurs.

The first feature film to be shown in multichannel sound. The original prints featured soundtracks that were recorded in a process known as Fantasound, a four-track directional stereophonic system that was invented especially to record the soundtrack for the film by RCA and the Walt Disney Studios technical team, led by William E. Garity. The Leopold Stokowski-conducted orchestra audio was recorded onto eight separate soundtracks (six channels recorded individual sections of the orchestra, the seventh recorded a mix of the first six channels and the eighth recorded a distant pickup of the entire orchestra), which were then mixed down to three tracks (left, center and right). The three music tracks were optically matted with a fourth control track (containing signal tones that varied the speaker dynamics) onto a filmstrip separate from the projector print. Over 90 speakers were used for the playback of the Fantasound audio during the premiere of the film on 12 November 1940. A more typical Fantasound setup used three speakers behind the screen and 65 others placed around the other three walls of the theater. However, Fantasound was discontinued due to the amount of sound equipment required and the time necessary to make the installation. The advent of

All prints made between 1941 and 1956 were re-mixed in monaural sound. The stereo was not restored until the 1956 re-release.