Yes, it is necessary to restart your Mac to make sure that the new changes take effect.
In conclusion, turning off Spotify on startup Mac is an easy but vital process that most Mac users should know. There are several methods that you can use to prevent Spotify from launching when you start up your computer. You can either use the app's feature to disable auto-start, stop it by modifying the login items list, or use Terminal commands. If none of the methods work, there are fixes that you can do to solve the problem. Lastly, hiding Spotify using the "Options" feature is also a convenient way to use the app when needed without crowding your dock.