Only the P1 controller has access to the Hotkey, so you can do any configurations for a game in the RGUI with a controller that has a hotkey defined (the one you chose when you configured the controller in EmulationStation). Make sure that controller has also an X button configured, otherwise you can't open the RGUI with the Hotkey + X combination.

To enable Multitap - and get more than 2 players working - you can do so from the Options menu, where you need to toggle on the Show other input settings, close the RGUI, then open it again and in the same Options menu, can set _Multitap_1 to ON and Pad 2 to Pad 4 to dualshock.
Since not all games support Multitap and enabling it globally would probably break input for it, make sure that you use the 'Create Game Options file' menu at the top of the Option list, then toggle off the Multitap option(s).

This one is resolved.
For everyone having the same issue.
In order to enable 3rd and 4th player through joysticks, you have to first install the ps3 or Xbox controller drivers through the retro pie setup menu.
Do not input any specific button configuration inside the per console config files.

If your aim here is to play these games with two controllers but still choose the player you want, try using the 2-player clones.

For example, tmnt2po lets each player choose which turtle they want to be.