Aurelio de Laurentiis must sell Napoli or Bari within the next two years.
The movie mogul took charge of Bari in 2018 when they were in Serie D and has rebuilt the club which saw them win promotion to Serie B this past season.
However, new laws made by the FIGC blocks one person or company from owning two clubs.
De Laurentiis saw his appeal against the rule rejected by the Federal Court of Appeal on Wednesday.
Bari are managed by son Luigi De Laurentiis, but their appeal was still rejected.
The family must therefore sell one of the two clubs by the start of the 2024-25 campaign.