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On ubuntu 11.04 I created a virtual machine with Windows 7. Now at /home/username folder I have both ISO image of Windows and a folder with the system created by VirtualBox. The question is can I delete ISO image and will it affect virtual machine?

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A virtual machine behaves similar to an actual machine

A virtual machine (VM) is a software implementation of a machine (i.e. a computer) that executes programs like a physical machine. -Wikipedia

So similar to an actual machine when you finish installing an operating system, you remove the disc, likewise, after you have installed Windows 7 you can remove the disk by

Select your Windows 7 VM > Machine > Settings > Storage > Select Disc Drive > Click on the CD icon > Remove disk from virtual drive

Then you can delete the .iso file and it will not affect your virtual machine.

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