Opal's hair length varies throughout the movie, from a short blunt cut, to a longer, and uneven cut, and then back to short.
Opal's hair changes from straight and flat to styled and blow-dried during the baseball game and party set-up scenes.
When it starts raining, at the start of the garden party, all of the lights strung around start to flicker and go out. Except when you see everyone standing at the house, the lights to the right can be seen still on.
The movie supposedly takes place during the summer somewhere in the deep south, but several characters' breath can be seen during the party scene.
When Opal and Gloria are walking by the mistake tree Opal's hair goes from being over her ear to behind it with no time between multiple times.
Gertrude, identified as a parrot, is actually a cockatoo.
As Opal was offering the Preacher a Litmus Lozenge when he told her "It's 2 in the morning, what are you doing up?", the lighting at the kitchen window shows that it's sunrise. It often gets dark at 2 AM.
During the scene where the animals get loose, the same event is used twice. When Opal picks up the black and white rabbit to place in its pen, it is shown once as a close up of her [at 33:41 from behind] and once from a distance [at 33:58 frontal]. (This can be clearly seen in the full frame version, but not in the wide screen version at 33:41 where the wide screen cropping of the spherical 35 mm frames crop out the body of the rabbit. Consequently, it is a goof in re-mastering the full frame version from the spherical 35 mm negatives rather than a goof in the wide screen film. Of course, it could be argued that the rabbit got out again and needed to be put into the pen a second time.)
The rat used for the beginning of the church scene is male, it then switches to being a female rat and back again between shots.
At the impromptu baseball game, the police officer notices that his underwear is showing and says: "don't look at that!" The actor's lips don't move. The dialogue was recorded elsewhere and added in.
The "mouse" Winn Dixie catches during the church service is a rat.