session_start() must be called on each and every page that wants to read and/or write data from/to the $SESSION array. It only needs to be called once per page and can be called in the main script or in an include as desired, as long as it's called before the first attempt to access $SESSION.
However, if by some chance you are including a file via a URL (instead of via the file system), then it would be processed as a stand-alone page so any session activity would be local to it. (Including via URL is potentially a severe security risk, however, so I hope you are not doing that.)