I think you are having an issue with the Anonymous Authentication Credentials on your website. Try this:
Open IIS Manager
Select your website
Open the "Authentication" pane
Select "Anonymous Authentication" and click the edit link in the Actions list.
Change the "Anonymous user identity" to "Application pool identity"
Alternatively, you could grant permissions on your web directory to the user that is specified in the "Specific User" field. I find it easier to just use one identity for a website.