Okay...
- You're sure the macro is actually running when you hit your hotkey?
- Have you tested with any apps other than Google Chrome?
Google Chrome has an ongoing bug that prevents accessibility from identifying it's front window.
Found Images Suddenly Broken in Google Chrome - #19 by ccstone
This bug should not affect your version that's looking at the whole screen though.
Big Sur's various Windows-Vista-like levels of transparency may cause issues, so try turning on “Reduce Transparency”:
-Chris