You can use = if you want, but then you need to reverse the then/else code.
If Me.txtEmployeeKey.Value = DLookup("[EmployeeKey]", "tblEmpKey", "EmployeeKey ='" & [EmployeeKey] & "'")
Then
MsgBox "Incorrect Employee Key Or Username already in use", vbOKOnly, "Incorrect Infomation"
Else...