In the season 1998–1999 Barzagli had his first professional football appearance with semi-pros Rondinella Calcio before moving to Serie B side A.C. Pistoiese in 2000/2001. The journey was short because the year later he rejoined Rondinella Calcio in Serie C2. He then signed Ascoli Calcio 1898 in season 2001/2002 and helped them to Serie B promotion. He remained in Ascoli for another year without further success. His first Serie A cap was won with Chievo Verona in the 1-1 draw at Brescia on August 2003. The year later the commanding defender was signed by Serie A outfit Palermo. Barzagli was bestowed with the captains armband after Eugenio Corini controversially left the Sicilian club in the summer of 2007/2008.

In 2008 Barzagli was in contract negotiations with ACF Fiorentina and was widely expected to sign. To everyone's surprise the deal fell through as Barzagli, a native Florentian, had already stated his eagerness to join. Barzagli's agent spoke to media saying: “The difference between what we were asking and what they offered was minimal – it amounted to £100,000. Andrea was willing to give up a lot of money to play for Fiorentina, but they didn’t take our requests into consideration". Instead Barzagli choose to join VfL Wolfsburg and the Bundesliga following fellow Italian and team-mate Cristian Zaccardo. It was later reported that the Italian international snubbed Fiorentina's five years £1.2mio contract over VfL Wolfsburg's 3 years £2.5mio offer. The transfer fee was reported to be at around €14mio.