Is the Enable Disk Option option ticked from under the iPod's Summary tab? If so, disable it to prevent other system applications such as your AntiVirus/Security software from accessing it.
Otherwise, reset the iPod with it still connected and then try ejecting it again.
Go to your computer's System Tray and try ejecting. If you can't eject, there is an application that is trying to access your iPod.
Go to http://technet.microsoft.com/en-us/sysinternals/bb896653.aspx and download ProcessExplorer.
Go to http://support.microsoft.com/kb/555665 and follow the instructions under Resolution.
Note that this is a good, general process any time you can't access any file.
In my case an update to RealPlayer installed a program called RecordingManager that was accessing my iPod.