Please excuse my skipping the rest of the replies - I thought it best to try and reply to your original question with a full answer rather than to each part independently.
From what I understand of the thread, you have made a backup of your course (a Moodle backup mbz file), downloaded that, and stored it offsite. You have then deleted the backup from within Moodle, but the file remains within the moodle sitedata directory.
To answer your original question: As long as you have that mbz file stored somewhere else outside of Moodle, and you backed up the course complete with all user content, you can restore the content safely, even once it has been removed from Moodle.
Normally when you remove all references to a file, it is moved to the trash folder. As others have pointed out, the Moodle cron system should remove unused files from the filesystem periodically. This normally happens once per day. It is possible to disable this, but it is unusual for this to be the case.
If a file is still somewhere within the nested filedir directory then that suggests that it is still in-use somewhere. This may be within another course, within a user's private files, or within a user's draft area. If it is in the draft area, then these file records are cleared about 4 days after that were last modified. Then they'll be moved to the trash directory, and removed shortly later.
Unfortunately, there is no easy way within the Moodle User Interface to find where references to a file are. I believe the Moosh tool has some functionality which can tell you; alternatively, you can run a database query along the lines of the following (you may need to change mdl_files to match your database prefix):
SELECT * FROM mdl_files WHERE contenthash = 'YourReallyLongContentHash';
If there are no records returned, then the actual file on the file system should be deleted within a few days. If there are records, then the file is still in use and will not be deleted.
As Howard mentions, we do recommend that you run cron more regularly than once per hour. Typically, we recommend once per minute to ensure timely delivery of all e-mail, reminders, updates, and notifications.
I hope this helps,