Factual error: During the motor boat chase through the canals of Venice, Dr. Schneider pilots the vessel between two ships, one identified as the John W. Mackay of London. Although the Mackay was commissioned in 1922, it was stationed at Halifax, Nova Scotia for service in the North Atlantic. It was deployed for the first time to the Mediterranean in 1942 - four years after the purported time of the movie.
Factual error: When Indiana's father uses the plane's gun, he shoots out the tail. This was not actually possible, as the planes had a safety mechanism to stop people doing this. In fact we can see that the Lewis gun he is using is mounted on a Scarff ring, which prevented the trigger of the gun being activated when the barrel was pointed at the aircraft's tail surfaces. (01:17:40)
Revealing mistake: When fire sweeps through the catacombs under the church, you can see that the "rats" are completely fake. At the far left of the screen (widescreen) you can see several fake rats just hanging on the wall and not moving at all. In the "special features" section on the DVD, Steven Spielberg admitted that the rats were fake - he couldn't bring himself to roast live ones so he could not make this scene appear realistic.
Continuity mistake: When the Nazi is strangling Indy with the chain, Indy looks down into the porthole of the tank and yells, "Dad!" Before he throws the gun down into the tank Indy's hat comes loose, but when the next shot is back on Indy, and he yells, "Dad! Dad, get out!" now his hat is back to normal.
Continuity mistake: When we first see the knight guarding the Grail, his cloak is tied off with a braided rope, which hangs down on his right side. It stays this way until he says "You have chosen wisely," where the rope has somehow shifted to his left side. The rope goes back to normal when the knight waves goodbye at the end of the movie. (01:49:10 - 01:54:05)
Continuity mistake: When the white robed men ambush the Nazis just after they blow up Salah's car, there's a shot over a Nazi's shoulder as he fires at one of the white robed guys running up a hill. The Nazi's machine gun is held straight out and doesn't move but the dust that flies from the squibs suggest that he fired as he moved the gun right to left which he did not do. The odd thing is that AFTER the squibs go off, the Nazi turns his gun to the left. (01:25:55)
Continuity mistake: When Indy takes four horses before battling with the tank, three are black and one is bay (brown with black legs, mane and tail). Sallah is riding a chestnut. When they ride to the Grail Temple, there are two black horses and two chestnuts. Where did the second chestnut come from? (01:28:00 - 01:39:25)