Does not equal is used to compare a Field to a single value (where if it matches, the record will be excluded from the set of results), it is used for fields of numeric, date/time, timespan and lookup fields (picklists, custom fields with a range of values, types, statuses, packages, projects etc.). It does not work for text fields, where you should use Contains and Does Not Contain instead.
Values can be either a single quoted, double quoted or unquoted value (useful for numbers etc.). Also, as elsewhere in the query language, where you use a string value, you can substitute a function call as well.
Priority is not low (unquoted value):
Priority != Low
Status is not First Draft (quoted value) for records of type requirement
EntityType = Requirement AND Status != "First Draft"
All records belonging to a single project called "Project X" (showing use of single quotes, but we could use double quotes instead if we wanted).
Project != 'Project X'
All records that were not created by me
CreatedBy != Me()