Short answer: Yes, おひとつ is perfectly natural.
As @Eddie Kal pointed out in the comment, お一人様 is also very common, as well as お二人様 and おふたつ. And I fully agree with @Eddie Kal that お is not used with more than 2 people/pieces. According to this site apparently it's not impossible to say お三つ, but it's not only unnatural, but also it's rather unlikely that the interlocutor understands what you meant to say. Other than these cases, I cannot really come up with a case where お is used with numbers.
For おひとつ and おふたつ, I don't think whether with or without お really matters (especially in the example you mentioned above, I don't really see a particular difference), but for お一人様 and お二人様, it sounds weird if you say just 一人様. If you are not sure, say 一名様、二名様、三名様 etc. These are Chinese numbers and by definition お is not used (if ever Chinese words are used in the honorific form, it would be with ご, although in this case for some reason you don't need ご).