Check the soldermask layer, esp in Eagle. You can easily tent/untent vias unintentionally.
Check paste layer to make sure you aren't putting paste on pads where you don't want it.
Check "unrouted traces" layer all by itself. Sometimes the ground plane doesn't quite reach areas, and you can't see it until you do this, leaving a 0.1mm gap between a pad and the ground plane. (Done this, and... uh.. where's the engineering inquiry when you need it?)
If you are doing any ongoing dev with the firmware, you can lay down a small area of solid white silkscreen for annotating with sharpie.
Beware that any insigificant but carelss things you leave on the board may turn into "engineering inquiries," otherwise known as questions. You don't want questions, because questions add time/days to your turnaround. If you're intentionally doing something that looks goofy, try to preempt a question by including that info when you submit the quote/RFQ.
Write down the dimensions of your board. You're going to fill out the form halfway and find out you don't know the dimensions. And the quote page will timeout.
If you're using a new manufacturer, be sure to put in multiple quantities, even some many times larger than what you originally anticipate. Some manufacturers have huge price changes over certain quantities. Once you find that magic number, it might save you tons of money in the future. 500 pieces might cost less than 400.