Are you sure you are adding the correct user? Have you checked to see which user is set for your applications app pool?
This error will also happen if it cannot read the file for some reason; such as the file is locked or used by another application. Since this is an ASP.NET web application you will want to make sure you are not performing any actions that would require the file to be locked; unless you can guarantee you will only have one user on your page at a time.
Can you post an example of how you access the file? What type of file is it? Code snippets will help you get a more exact answer.