Will using accessories while driving on the highway hurt mpg?

Shouldn't be a problem. The 12V battery charging is administered from the HV battery and doesn't command the engine directly anyhow. Any time the car is in "ready" mode (the equivalent to "on" in any normal car) the battery is being charged to roughly 14.6 V whether the engine's running or not. The state of charge for the traction battery will kick the engine in if it gets too low but this is all transparent to the 12V battery since it's bathing in the charging voltage all the while.
You'll see people having problems with their 12V batteries but it's not a reflection on the car's charging of it.
It's because
1) There are no day-to-day cues that suggest that the battery is nearing the end of its life (since it doesn't crank the engine--it only powers up the computers).
2) The smart key system exerts a very small parasitic drain while the car is parked.
3) It's a very small battery by modern car standards.

 

The car will run the engine whenever it decides it's needed for help in propelling the car and/or for charging the traction battery.

 

BUT, the energy has to come from somewhere. There is not free lunch, remember?

Still, the current draw will generally be minimal most of the time. Once you start drawing the equivalent of 800+ watts the effect on fuel economy is noticeable.

Measuring the draw from the AC unit during the summer we often see 1500-1800 watts of current draw until the cabin is cooled enough to allow the compressor to wind down. During this time you can observe a 7-10mpg instantaneous drop in fuel economy.

 

Big-nice person stereo: 2,000 Watts. Prius moving at 70 MPH: 60 HP = 40,000 Watts. You do the math :_>

Just remember to keep the car fully ON ("Ready") whenever you're using the stereo and the batteries will be fine.

 

Not to mention that the 2,000 watt figure has to be a peak power rating. 2,000 watts worth of continuous output would purée your organs (or at least your brain) before too long.

 

In theory, every bit of power used by a non PIP comes from gasoline, so yes it lowers MPG.
In practice, you can't measure it. (always leave your Prius in Ready when using electricity)