"They are not little girls."
Translation:Ony nejsou malé holky.
No difference in formality. Negligible difference in meaning: If you include "ony", you are showing that whatever you are talking about is of the feminine (or masculine inanimate) gender. If you skip the "ony", you are not revealing the gender. If you were talking about boys (kluci), you would not use "ony", but either "oni" or nothing.