Litherland REMYCA thought they had set up a tie with Leek Town after beating Charnock Richard 4-2 in the extra preliminary round back on August 11, only for the clash to be postponed pending an investigation.
As it turned out, the ninth-division outfit had fielded a player who had been issued with a one-match ban for his failure to pay a fine at his previous club against Charnock Richard, with that ban carrying over following his transfer to Litherland.
A club statement read: "Despite what we considered a strong case and representation at appeal hearings, today the FA have considered their case regarding the ineligibility of the player as proven.
"This is a massive blow with numerous implications, including financial issues as part of the penalty includes removal of prize money and future gate and match day income.
"The sanctions for a minor administration technicality are considerable, but we must dust ourselves down, learn from the experience and come back stronger."