Error 500 on first request for file

Solution 1:

In order to get some more details, I recommend that you enable "Failed Request Tracing Rules"

First, go to "Edit Site Tracing" and check it as "Enabled" note the location of the trace files, inside that location, you will find a directory with the site's ID.

1. Go to IIS Manager -> Sites -> your site.
2. Double click on "Failed Request Tracing Rules".
3. In Actions (right side) select "Add..."
4. Choose "All Content (*)"
5. Select "v" for Status codes(s) and type "400-599" (that will cover the error ranges, including files-not found), click Next
6. Select all the providers, and continue to Finish.

After the first request, on the Server, go to the location of the Trace files, and open it in Internet Explorer.

I hope that will help you find the fault module, or where the problem is really at.

Let me know if you need anything else, Idan.