Di Biagio signed for Ascoli in November 2006, but the bid was not considered to be valid by the federation, since the player was not released for free by Brescia before the 30 June deadline. The bid was therefore postponed until January 2007, and in the meantime Di Biagio went on training with Ascoli, and played from November to December with Promozione club Polisportiva La Storta from Rome, coached by his friend and former Dundee and Lazio footballer Alessandro Romano. Di Biagio played his first Ascoli match on 14 January 2007, against Cagliari. He collected only 7 appearances that season, scoring two goals in 523 minutes. Ascoli finished second last in Serie A that season and were relegated to Serie B. retired at the end of the season, and returned to Polisportiva La Storta as a youth coach.