Yes, they can be overwritten - but the problem you are having is that the code you wrote to write teh file in teh first button press (and probably the second as well) does not clean up after itself as it should.
If you open a stream (or other file related object) for writing, and do not Close and Dispose of it when you are finished, it remains open until the Garbage Collector decides it is no longer needed or your application ends. This could be next week for all you know!
Either use a Using block:
Using MyFile As New FileStream()
Or call the Dispose Method when you are finished with writing the file.