Revealing mistake: While Indy and Marion are in the Well of Souls, and they encounter the snakes, Indy falls to the ground only to be confronted by a hissing Cobra rearing its head. Look carefully and you'll see the snake's reflection on the safety glass between it and Indy. Briefly you can also see the torch's reflection while he's waving it around. You can also see Marion's reflection when she falls in later on and is face to face with the cobra. [This has been corrected in the new DVD set. You can only see it if you have the original VHS. I think it's visible in the "making of" on the DVD as well.]
Continuity mistake: When Indy goes to save Marion in the tent he removes the handkerchief gag. But when he decides to leave her there, he puts the gag back on and some of her hair gets caught underneath the gag on one close up shot from the left. But when the camera goes to the back of Indy, her hair is no longer caught underneath the gag. (00:56:00)
Continuity mistake: Near the beginning of the movie where Indiana is running away from the tribe of South American Indians after Belloq robs Indiana of the gold statue, in one shot you can see that the Indians are only a few meters behind Indiana, but in the next shot, they are at least 50 meters behind. (00:11:20)
Plot hole: When Indy and Sallah visit the gentleman who translates the staff headpiece for them we hear the staff should be "six cadams high." Indy replies, "About 72 inches." Then, turning the headpiece over, "Wait - take back one cadam to honor the Hebrew God whose ark this is." So, about five feet now, right? But, when Indy goes to the map room the staff is much taller than him. If the staff is about five feet high, Indy would be around three feet tall. (00:48:25 - 00:53:05)
Revealing mistake: When Indy fights the driver of the Nazi truck, he gets thrown through the windshield, and eventually ends up underneath the truck. When he is climbing underneath the truck, heading towards the back of it, you can see the trench that was dug down the middle of the road to provide the stuntman (this being one of the few stunts that Ford didn't do himself) with the extra room needed to do this feat. (This is backed up by DVD commentary. The director stated that the truck was too low to the ground so they had to dig the trench in order for there to be enough room for the stuntman). (01:27:50)
Revealing mistake: After Indy gets behind the wheel of the truck containing the ark, he jerks the steering wheel to the right to run the pursuing jeep off the road. He then jerks the wheel to the left to hit an upcoming motorcycle. In neither case do you see his body sway with the presumably drastic swerve of the truck. Apparently this was filmed in a stationary vehicle, or one in tow that was fitted with a prop steering wheel.
Continuity mistake: In the Himalayan bar scene when the sherpas leave, Marion starts to pick up the shot glasses and puts them on a tray - there's a large group left at one end of the table, one in the middle, and two by a bowl at the other end. When she returns for the rest, the two by the bowl have vanished. (00:26:20)
Revealing mistake: When Indy jumps across the pit to pursue the spy that took the idol, he barely makes it across, grabbing the ledge on the opposite side with his hands. When he pulls himself up, he kicks loose a rock and the entire "solid" rock wall shakes like it's nothing more than a foam wall.
Continuity mistake: When Belloq is talking to Indy and Marion in the Well of Souls, he's shown holding his hat with his right hand. But in the only over-the-shoulder shot (looking into the Well of Souls) the hat is laying on the ground and Belloq tosses a stone down with his right hand. Then the hat is back. (01:10:35)
Continuity mistake: Before the shoot-out in Marion's bar, before the Nazis enter and ask her for the amulet, she's sitting alone at the table. She pulls the amulet out of her shirt, the chain still around her neck, and stares at it in the candlelight. The next moment the chain is not around her neck and she hangs the necklace onto the table centrepiece. (00:28:20)
Factual error: When Indiana Jones is fighting the big German by the airplane, the German punches Indiana Jones on his right cheek. Indy's face and body are both knocked down to the right, in the direction of the punch. When you watch this, you can see it's impossible to be knocked down in such a way. (01:17:55)
Factual error: It should be a tricky task to put an object the size of the Ark on board a German VIIC submarine; perhaps you can find enough place inside but how do you manage to introduce that bulky case through the narrow hatches of the sub? Even the torpedo load hatches were just a little more than 21 inches in diameter.