m not really sure what youre getting at but I`ll take a stab at it since no one else has answered, yet.
What you want to do is decrypt all the files if you plan to copy the entire DVD or else decrypt just the movie vobs along with the ifo file if you want just the movie without any of the extras. These should be placed in one folder. I just name the folder after the movie. When you start DVD2one, you point to that folder and then select a folder for DVD2one to place the new files. This folder I name VIDEO_TS. After you have burned your DVD you can delete this folder. Using this method you will be less likely to add files to a folder already containing files. You do not want to contaminate new files with old files so I`m guessing the answer to your question is "Yes" you need to delete the old files from the last job before adding files for the new job.