Found the problem. The back template I used for my card type had <!–{{Audio}}–> instead of just {{Audio}}, so amending that fixed it and audio plays again. My card templates came from the itazuraneko guide: anki guide
It had nothing to do with associations between cards and files, and the unused files were presumably from shared decks I had used in the past, but somehow the files remained even though I deleted those decks long ago (just guessing). I have now deleted those files, nothing shows up as unused or missing, and everything is working.