Numerous time in the film the animals walk from shade into sunlight without their eyes reacting.
When Mufasa has to teach Simba a lesson it's still very light all around. By the time Simba walks up to him it's almost dark.
Technically a lion wouldn't be able survive on just bugs, no matter how much protein they have. Simba at some point realistically would have become malnourished and may have been forced to eat other animals.
Lions live in matrilineal prides, so Nala should've become the Lion Queen.
Lion prides are made up of related females and a few breeding males at most. Territories are passed down through the female line.
When we see young Pumbaa in flashback, he is clearly modelled on a juvenile wild boar rather than a warthog.
Simba and Nala would never mate. Given the pride dynamics, there's no way that Simba and Nala aren't related. At the very least, they're cousins, but more likely they're actually brother and sister. So they wouldn't be feeling the love tonight.
Microphone peaking can be heard during vocals at several points. Examples include the end of "one and all" in "The Circle of Life" and the start of Simba's "All of this?" in the "everything the light touches" scene.
Before the stampede, several wildebeest are shown grazing on grass-less land.
The final note of the "free to do it all my way" line in "I Just Can't Wait To Be King" is pitch-corrected to the wrong note.
Adult Simba roars atop Pride Rock, but the sound he makes is the typical Hollywood beefed-up roar rather than an actual lion vocalization. He also does the stereotypical wide-mouth yelling posture commonly associated with roaring, but actual full-blown lion roars are produced with the mouth in an almost closed position similar to a howling wolf. However, the roars used are NOT tiger roars, as is often stated. Tiger roars are actually higher-pitched than those of lions.
Near the start when Mufasa says "Let me explain," to Simba, his mouth doesn't move.
Rafiki's design seen throughout the promotional materials firmly establish him as a mandrill (as opposed to a baboon), resulting in him being very out of place as mandrills hail from the rain-forests of West Africa, as opposed to the East African Serengeti.
An African gray parrot is seen hitching a ride on an elephant's tusk in the international trailer despite not being native to southern Kenya.
Timon being a meerkat flies pretty heavily in the face of the species' actual bio-geography given that meerkats are endemic to the Namib and Kalahari desert regions of southwestern Africa.
A group of gemsbok (Oryx gazella) are featured in the first teaser trailer even though the species is only found in southwestern Africa rather than the East African setting of the story. The similar and related East African oryx, specifically the beisa oryx (Oryx beisa beisa), would've been a more appropriate choice.
Scar's cover-up of his murder of Mufasa leaves a gaping hole: he sends Zazu to get the pride for help, and after the stampede, claims to the pride that he didn't reach the gorge in time to help Simba and Mufasa. Zazu is implied to have been exiled from the pride after Scar's take-over (given how the hyenas regularly try to eat him when he shows up), but considering Zazu still clearly regularly visited Pride Rock to relay information, it's a wonder how Scar's lie about not being able to make it to the gorge didn't get exposed by Zazu.
During Scar's reign, the Pride lands presumably suffers a drought. With that being the case, it would be impossible for the lions to live without water even for a few days.
When Simba and Mufasa are out patrolling, Zazu reports "Hyenas in the Prideland" and informs Mufasa that Sarabi is "leading the charge". Mufasa tells Zazu to take Simba home, where Sarabi is calmly washing cubs with the other mothers.