Not to put too fine a point on it, but you are a scientist – this is easy to determine experimentally.
Rosetta definitely allows you to use command line flags and flags files simultaneously. Our integration testing system takes advantage of this, for example.
If you specify multiple arguments for a command, it just uses the last one specified. If it is a vectorized flag (takes multiple arguments) it may append instead.
It appears to parse options in the order they are specified, so the @file is processed after options that are before it and before options that are after it.