No. A workgroup file applies to the Access session, not the database.
There is always a workgroup file used, even in unsecured mdb files.
Out of the box, Access uses system.mdw, a workgroup file that ships with it.
It silently logs you in as the 'Admin' user. Every system.mdw has the Admin
User (and the Users Group) in common. So anyone can open your unsecure mdb,
since 'Admin' (the common user) owns everything and has permissions to
everything.
When you secure a mdb, you create a new workgroup file, and using it, you
remove all permissions for the Admin User and the Users Group. The
permissions are stored in the mdb itself. When someone tries to open it
using system.mdw, they can't because 'Admin' doesn't have any permissions.
When you start Access, it will use the workgroup file that is set as the
default. Normally one would leave the default set to system.mdw, and use a
desktop shortcut to launch secure databases. The desktop shortcut would use
the /wrkgrp switch to specify a secure mdw, which would over-ride the
default system.mdw for that session of Access. Opening Access without the
shortcut, would continue to use the system.mdw as that is set as the
default.
I suggest you use the workgroup administrator to set system.mdw as your
default. Use a desktop shortcut to launch secure databases.