The two mosquitoes trapped in the light of the bugzapper ("Frank, don't go towards the light!" "I can't help it - it's so beautiful!") are the voices of the co-directors, John Lasseter and Andrew Stanton.
In the bloopers shown at the end of the film, Princess Atta is shown cracking up during her scene with Hopper, ruining take after take until Hopper goes to his trailer in frustration. This is a spoof of Julia Louis-Dreyfus being known to do the same.
The tunnel in a tunnel joke was made in reference to Steve Jobs' (CEO of Pixar and Apple) plan for Apple's "store in a store". Also, the hand gestures made by Hopper were modeled after gestures made by Jobs.
For the 1.33:1 video transfer, rather than pan-and-scan the original 2.35:1 theatrical image, Pixar actually re-used the original computer images, re-framed some scenes, and even to the point where they'd place characters to a different spot in the scene to fit into the 1.33:1 frame.
When Flik is in the city, a cereal box can be seen over his right shoulder with the first half of the code written on it appearing as "A113", a reference to the animation room at California Arts University where many Pixar artists attended.
Editor Lee Unkrich revealed they considered calling the film "Bug Story," but coming on the heels of Toy Story (1995), they were worried that people would expect Pixar to use the "____ Story" naming convention for all their films.