I am making a subfloor for a new bathtub on a 2x4 frame, 16" center and 3/4" plywood decking. Should this be sufficient to support the tub, (~500 lbs. completely filled)?

Thanks

I would have to say so. The weight of the tub will be distributed through the floor area.
Unless the tub has 4 legs for support, you should then add additional 2x's under where each leg will rest.

What is the subfloor frame going over, existing wood floor or concrete?

The floor is going over concrete and is intended to support a whirlpool tub.

Thanks!

You'll be fine.

You are quickly becoming my hero! :-)