Twonky is a strange beast. You might find that reading my FAQ helps:
Or this discussion on where to put your media:
My first suggestion would be checking that Media Serving is enabled on the Shares|Public|Share Profile page.
For some reason, WD chose to make this ‘off’ by default, but enable ‘Media Streaming’ by default. Both need to be on to get Twonky to catalogue and stream media…