We can mix up both C# and VB.Net in App_Code. For that purpose we have to perform these two steps:
1. Create two subdirectories in App_Code folder one each for C# and VB.Net. Let suppose i have created directory named "VBCode" for VB.Net and "CSCode" for C#.
2. Modify the complition section of web.config file like this

<compilation>
     <codeSubDirectories>
            <add directoryName="VBCode"/>
            <add directoryName="CSCode"/>
      </codeSubDirectories>
</compilation>