Application for Locking folders

The best way is to create a disk image using Disk Utility
You can secure it with a password
Store your files there, but make sure the password isn't stored in keychain

Woof, Woof - Dawg

 

Edit: beaten by the 'Dawg.

Not quite what you want, but built into the system:

• Open Applications->Utilities->Disk Utility
• Choose "File->New->Blank Disk Image..."
• Set the name, size, format, partitions, etc. as you like.
• Choose some form of encryption
• Choose "sparse disk image" to save space
• Click "Create"
• Enter a password but be sure to uncheck the "Remember password in my keychain" option.
• Click OK.

This will create a very secure disk image that, when double-clicked, will ask for a password before mounting. If you don't save the password in your keychain, this is very very secure. So secure, in fact, that if you forget the password, all of the data is lost.

Once the image is mounted, drag things to and from it, then, before someone else might use the system, just eject the image. You'll need to double-click on the sparse image file again and reenter a password each time to open it, but it'll stay open until you eject it.

As a plus, you can back up the sparseimage file, copy it to a flash drive, email it, whatever. It is completely secure if it falls into the hands of someone who doesn't know the password. Well, assuming you pick a non-trivial password.