Revealing mistake: When Becky sees the spider in the shower, there are two instances when you can see the bodystocking she's wearing, covering her breasts, to preserve her modesty, most noticeable when she screams.
Revealing mistake: When the scientists inject the mouse with poison, they put it in a jar. The, after they place it down you can see someone's hand swap the jar for one containing a dead mouse. You can even hear it.
Trivia: Near the start of the film, when Atherton is examining the spider after returning from the valley, several butterflies are seen pinned up in front of him. Several of these are not South American butterflies - the top left one (blue with long tails) is Papilio Ulysses, a species of tropical Australia, and the big green-and-black one near the bottom right is Ornithoptera priamus or O. euphorion. Whether this qualifies as a mistake, I don't know - Atherton is supposed to be an expert, so they may not be the most geographically accurate 'colourful' butterflies to use.
Trivia: Since spiders really aren't a trainable type of animal, the filmmakers used hairdryers to blow on them, in order to get them to move correctly.