What you may check is whether any VM still has a reference to the datastore (e.g. a snapshot or ISO image attached to the virtual CD-ROM drive). You can see which datastores are "in use" by a VM on it's Summary/General tab. Another option to check whether a datastore is still referenced is to use RVTools (http://www.robware.net/).
Since you mentioned HA, it's also possible the datastore is in use by the HA datastore heartbeat. You may check this in the HA settings.
André