Is the ISO still attached to some VMs?
When logged into the ESXi command shell on the host, use 'lsof | grep <name-of-iso-file>'.
Then, use the process id number in the left most column to find the matching virtual machine that's using this image.
For example: 'ps -c | grep <process-id>'
Finally, in the guest VM configuration in the ESXi host UI or vSphere Client, edit the virtual machine settings.
Untick the 'Connected' box next to the CD/DVD drive.
This will unlock the file and you can remove it.