How can I make a two voltage source, 4-layer PCB in Eagle?
Spacing isn't important with such a close voltage difference. 0.3mm should be more than enough although I would go larger just because. It makes a nice visual distinction between board areas when it's larger.

I had 5mm between 3.3V and 120V on one my board, and about 0.5mm between 15V and 120V in some places (which was probably too small to be safe, but it was a special gate-drive circuit and was isolated from the user) but still encountered no issues. If you want to use official standards the term you need to Google is "PCB creepage distance".

I assume that's an H-bridge? I guess there's a chance something could spark over if the inductive kick was large enough but you should have flyback diodes anyways to deal with that (or the MOSFET body diode but that is nowhere near as good).