Can I use the same keystore file to sign two different applications?

You can use that keystore for any number of applications.

No need to generate a new keystore.

I'll make a counter argument to the consensus answer so far.

I agree that for most app authors most of the time, sharing the same keystore/certificate/password between your apps will work fine. The critical thing is to use "the same certificate throughout the expected lifespan of your applications" so the app can upgrade itself.

But I can think of one very good reason to have separate keystores for separate apps or families of apps. If you think you might ever want to sell an app to someone else for them to publish as an upgrade to the original, you'll have to share your one-and-only keystore and password with them to do so. Probably not a huge issue but a bit of worry to you and, perhaps, a due diligence issue to a big-enough buyer.

Also, I really don't read the same line in the documentation the same way as @ol_v_er does. I think the current line:

You should sign all of your apps with the same certificate throughout the expected lifespan of your applications.

(note the lack of a comma in the current version) is simply emphasizing that the 'lifetime' recommendation applies to all apps, not actually directing you to use the same certificate for all of your apps.

The official documentation tells us:

In general, the recommended strategy for all developers is to sign all of your applications with the same certificate, throughout the expected lifespan of your applications. There are several reasons why you should do so ...

https://developer.android.com/studio/publish/app-signing.html#considerations

So yes, try to sign all of your applications with the same certificate.