No - once a file is saved to a backup drive, you retain the history of how that file looked.
Similarly - an exclusion only changes what is considered to be backed up going forward, the exclusion does not apply retroactively.
You would need to enter Time Machine and explicitly delete all backups of the excluded folder to have that cleanup of backed up files be surgically removed leaving the rest of the back up intact. The gear control allows you to select delete all backups of the selected file(s) and folder(s)
There’s also hope we can use a command line tool to script the gear UI removal of a specific file / folder from the command line.
The only exception is if Time Machine runs out of space to make a new backup. Then and only then, when the Time Machine drive becomes full, backup copies of a file will potentially be erased. The oldest backups get purged first, so most people can get back the file from backups weeks or months ago even if the one from two years ago gets deleted.