My guess is that your steps/unit is wrong.
If it's not already, set your Native Units to inches. If it was mm, close and restart Mach3. Then, make sure that you're in inch mode. Then, move and see if it's moving the correct distance. If it appears to be in mm's, then most likely your steps /unit needs to be changed.
If you originally set up the machine with the native units set to mm's inadvertently, it's possible to set the steps/unit in inches, and it will appear to be correct, until you run g-code with a G20.