You mean synapse? Yes, sure.
No, I mean an app has to be installed on mydomain.com for the well-known to be served. If there is no app, nothing is served.
The matrix domain setup can be tricky, if you are not using the defaults suggested in https://docs.cloudron.io/apps/synapse/#post-installation . For this reason, can you explain your setup again and also what matrix id you are trying to achieve? List the domains clearly and if you have done the 3 steps listed in the doc link.