The 31-year-old was found dead in his hotel room having suffered from a cardiac arrest, just hours before he was due to lead Fiorentina onto the field against Udinese in Italian football's elite Serie A league.
Teammates, fans, politicians and former rivals alike came together for the service at the Basilica di Santa Croce in the heart of the Tuscan capital to remember and celebrate the life of the Italian defender and grieve his untimely passing.
Fiorentina scarves and banners were held aloft, branded with messages of love: "Per Sempre Nostro Capitano" -- "Forever our captain".
Many of the most famous faces from Italian football were present, including the full Fiorentina squad, Nice forward Mario Balotelli, former AC Milan attacker Marco van Basten, Inter Milan manager Luciano Spalletti, and legendary defender Javier Zanetti, as well as former Italian Prime Minister Matteo Renzi.
Members of the Juventus squad and coaching staff arrived, too, having flown straight from their Champions League Round of 16 tie against Tottenham at Wembley Wednesday evening.
Juventus captain and former Italy skipper Gianluigi Buffon was in tears as he entered the basilica, while defender Giorgio Chiellini -- another former teammate of Astori -- was also visibly emotional.
In the aftermath of Juventus' second-half comeback against Tottenham, Chiellini dedicated the win to Astori, adding: "Not only this match, but every mind on this day."
In his post-match interview Wednesday, the 33-year-old told BT Sport: "He is always in our hearts, and tomorrow we go to give the last 'ciao' together with our teammates."
UEFA announced Monday that all Champions League and Europa League games this week would hold a minute's silence in memory of Astori, and Juventus fans at Wembley held aloft the message "RIP Astori."
All of Sunday's Serie A games were postponed in the immediate aftermath of the tragic news as tributes poured in from around the world.
Chiellini admitted that even by Wednesday, it was hard for the Juventus players to focus on the match, adding: "It is very difficult but we have to continue our lives and I think for us it's important, for the national team in our hearts to always bring his positive emotion -- he always smiled."
Juventus, the 2017 Champions League finalists, recovered from a 1-0 halftime deficit with two goals in three minutes from Gonzalo Higuain and Paulo Dybala to go through to the quarterfinals, overcoming Tottenham 4-3 on aggregate.