Yes unless you can move it to a different I/O port to avoid a conflict. Or use one for joysticks and the other for MIDI, which use different ports. I'm not sure whether the MIDI portion still works if you disable the joystick, but it might.

You might also have issues detecting the OPL chips if they both have FM synths as they will both try to respond on the same I/O ports. However I tried this years ago and got FM synth coming out of both cards at the same time in all the games I tried without any problems, so possibly only the manufacturer's own apps might give you issues.

One of the cards is a card with Yamaha ymf724F-V chip that has joystick port and the second one is Sound blaster audigy 2 zs that has joystick port header on the card. I just got the audigy card today. Not really sure if the audigy joystick header only works as joystick port or does it work with midi as well.

The Audigy is a "modern" card so the joystick port is unlikely to work in DOS. Even the earlier SBLive needed special emulation drivers to work in DOS, which I think they stopped supplying with the Audigy. So as far as DOS is concerned, you will only have one card - the Audigy will be invisible.

In Windows, I'm not sure how the modern joystick port works but I suspect it won't use the same fixed resources they used to. So you will probably find that both joystick ports will work at the same time.

Sorry I was assuming when you said two sound cards that they were both older DOS-compatible cards. Since the Audigy isn't DOS-compatible (as far as I'm aware) then there should be no conflict between the two.

I guess I assumed it too that because yamaha card setup sets the dos joystick port, having a second pci card with joystick port would interfere. I just generally thought joystick ports were meant for dos, because by the time audigy 2 zs was released, I bet pretty much all joysticks meant to be used in windows were usb joysticks.