I have been having this issue for months where PNGTuber Plus will work fine and register the microphone well, but after a few minutes it just stops. I thought it might've set itself to go to the other audio options and so I isolated my mic and set my headset to stop registering itself as a microphone in my System Settings, but unfortunately even though the mic is isolated and set as default on startup of PNG Tuber Plus, after the few minutes it just stops registering the option entirely as it shows it as an option, but doesn't respond when selected but on some occasions it does.
I'm sorry for having this pop up more but any other PNGTuber Software ain't an option unfortunately due to complications and setup.