Can I use "os senhores" to address an audience of ladies and gentlemen? If my audience consists of men only, I can surely say "os senhores". What about the common situation where both women and men are present, can I say "os senhores"? Or would I have to say "os senhores e as senhoras"?
The grammatical question is very clear: in Portuguese feminine is the marked gender, so it will refer to feminine entities only; the masculine is the unmarked gender, so it can refer to a mixed group of feminine and masculine entities. Hence senhores, like todos, cidadãos, companheiros, etc. can be used to refer to or address both a group of men and a mixed group of men and women.
This is as far as grammar goes. Then there is the question of social acceptability. There have been of late voices clamoring for an “inclusive language,” that would refer to or address a mixed group of men and women with senhoras e senhores, cidadãos e cidadãs and even todos e todas. Some politicians have adopted this language. Some administrations, such as that of Rio Grande do Sul, set up committees to draft manuals for non-sexist language. My hunch is that in Portugal and, judging by my Brazilian friends, in Brazil hardly anybody cares about “inclusive language.” Of those who, occasionally, care it is more often than not to ridicule it. See this article in Gramaticalhas and this one in Veja.
That said, when addressing an audience of both sexes, say when you’re giving a speech at a conference or a dinner party, a time-honoured form of address is senhoras e senhores:
Senhoras e senhores, é com grande prazer...
This is equivalent to the English formula ladies and gentlemen. In fact, when appropriate, it can even be senhoras e senhores, meninas e meninos. Think of a circus. As an addressing pronoun though, I find a senhora e o senhor or as senhoras e os senhores rather cumbersome. Imagine asking a couple:
Então, e a senhora e o senhor donde vêm?
I'd simply say, então, e os senhores donde vêm? and I would have grammar on my side. If you’re wary of offending anybody, Portuguese lets you omit the addressing pronoun in most, although not all, circumstances. You could say to a couple for instance:
Ora muito bem, façam então o favor de me acompanhar.