Weird things like this tend to be caused by multiple firewalls.  Any chance you disabled Windows Firewall/Defender but there is also Webroot or McAfee installed?  If one username works, all should but if you are logging into Windows one each Workstation for each Sage 50 username, then you are adding complexity in determining the problem due to Windows security (I don't know if firewall settings are per user).

I can't tell from your message if you are referring to Windows users or Sage 50 users having problems connecting.  Let's run through some questions.

1. On just one Windows Workstation login, can the program open the sample file on each computer?
2. On just one Windows Workstation login, can the program open the live file copied to the c:\Sage50Data folder on each computer using each users' Sage 50 username?
3. As you probably know 1 and 2 tell you the program is operating fine on each workstation and your company file is fine (added this for others reading and wondering the reasons behind the questions).
4. On just one Windows Workstation login, can you create a text file in the same folder as the *.sai file from each computer?
5. On just one Windows Workstation login, can you create a text file in the *.saj folder from each computer?  Delete it afterward but be very, very careful not to delete anything else.
6. 4 and 5 tell you Windows security is set up to allow users to edit the database.
7. On the server, does the Connection Manager show that it is running and if any connections have been made?  If the file is closed, then no connections but the Connection Manager should always be running.  You can use the program on the Start Menu, the icon in the systray or check in the Services module.
8. Is Sage 50 full program installed on the server?  If so, try to open the file from the server (same idea as #2 above).  If this works then the problem is likely the inbound firewall settings.  You may have to Allow an app through the firewall (usually the mysqld.exe and SimplyConnectionManager.exe programs - Private or Domain)
9. Try opening the program from a workstation again and check the Connection Manager window on the server.  Does it show any connections?  Does it show them, then they disappear in about 20 seconds?  Probably a firewall issue.

The Windows Event Viewer may shows something.  Other than this, I can only suggest going through the Knowledgebase articles to see if there is something there that can help.  There is one for allowing programs through the firewall.  I've never had to add as many programs as listed in the article.