Your replacement string is empty. Try:
string output = Regex.Replace(Usernames, "''''", "\"");
[edit]
Here is the actual code I used:
string Usernames = "''''testing1'''',''''testing2'''',''''testing3'''',''''testing4'''',''''testing5'''',''''testing6''''";
Console.WriteLine("Input: {0}", Usernames);
string output = Regex.Replace(Usernames, "''''", "\"");
Console.WriteLine("Output: {0}", output);
And here is the resulting output:
./test
Input: ''''testing1'''',''''testing2'''',''''testing3'''',''''testing4'''',''''testing5'''',''''testing6''''
Output: "testing1","testing2","testing3","testing4","testing5","testing6"
[/edit]