Hi Beth,
Happy New Year!
One thing that did change a few versions back is that the Web.config file has runAllManagedModulesForAllRequests="true" on the <modules element in the system.webServer section.
This is needed to support extensionless urls but if you are not using extensionless urls you could try removing that and see if it changes anything. With it true then .NET is involved in all requests including those for static files like css and js and if not true then those static files are just served by IIS. So removing that should eliminate .NET code from the equation for requests for those files and then we can determine if the error is happening in .NET code.
Do you have any custom http modules plugged in in Web.config other than the ones included by default in mojoPortal?
Are you accessing the site by ip address as your screen shot indicates or is that happening only for those css and js files?
Also from the screenshot you sent it seems you have
<add key="CombineCSS" value="false"/>
in your user.config file so it is rendering static links to all of the CSS files instead of combining them into one request. I would highly recommend changing that to true. Other settings such as cache settings have no effect when that is false because with static urls the browser will cache them whereas when they are returned from our csshandler.ashx we can specify the cache settings.
Best,
Joe