Question: Salve! Having created a web-application under the Sharepoint (2010) Site's directory in iis7, I now need to use the assemble microsoft.sharepoint in my web-application. How can I reference that? 

I can confirm that the reference is being inherited from the parent's (sp) web.config, because if I add the reference, asp.net complains that there is a duplicate entry.

What can I do?

Answer: 
My recommendation would be that you shouldn't.

You should start from scratch again and develop your Web-Application to be deployed below the \_layouts folder in SharePoint using a WSP package and all of your code should be in code behind which is compiled into a dll deployed to the GAC.

You can technically get your solution to work, but it's a hack, that won't work when an extra server is added.
Add the SharePoint.dll as a reference in your project:
\Program Files\Common Files\Microsoft Shared\Web Server Extensions\14\ISAPI\Microsoft.SharePoint.dll

I assume this is SharePoint 2010.