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Is there a way / tool to lock folder and all files inside for specific time to prevent any other program on PC to perform any actions? Something like anti-Unlocker tool for Windows.

I use Windows 7. The goal is to secure data while running some unsafe and not tested software.

I think it is have something to do with handles which acquire access while performing operations.

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Sure there is. Why are you asking? Depending on what you want to do, you could write code, a script, a batch file, any number of things. You need to tell us what OS you're dealing with, and what your ultimate goal is, before we can give you a decent answer. – user3463 Apr 26 '12 at 22:25

The easiest solution by far is to run the untrusted software using an account which doesn't have write permission on the sensitive files. This can be done simply with a Deny Access Control List entry. This will override Unlocker-style actions from the untrusted software (since (A) the file isn't locked and (B) the software wouldn't have permission to unlock it anyway).

If you want it timed, use a scheduled task to remove the ACL using CACLS.EXE.

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