Father Luigi Ciotti, the president of Libera, an association of civil society groups which organised the march, told the crowd: "A day like today is meaningful only if we keep fighting the other 364 days of the year."
Libera carries out a number of activities aimed at tackling organised crime, including acquiring farms and buildings confiscated from the mafia and using them for social good, such as schools and drug rehabilitation centres.
Italian police have cracked down on the Sicilian mafia in recent years with the arrests of several prominent mafia bosses.
But Italy's four biggest mafia organisations - Calabria's 'Ndrangheta, Sicily's Cosa Nostra, Naples' Camorra and Puglia's Sacra Corona Unita - are still believed to account for a large portion of the country's economy.
Roberto Maroni, Italy's interior minister, has said that the 'Ndrangheta alone, now considered the most powerful of the criminal organisations, makes $61.6bn a year through its hold on the European drugs market.
"The mafia and the Camorra are not eternal. They can be beaten"
Antonio Bassolino, Compania president
Italy's intelligence services warned earlier this month the global downturn was giving the mafia the chance to tighten their grip on the economy as they use proceeds from illegal activities to buy stakes in the retail, tourism and real estate sectors.
Antonio Bassolino, president of the Compania region that includes Naples, called for more police and judicial resources to help combat organised crime.
"The mafia and the Camorra are not eternal. They can be beaten," he said.
Roberto Saviano, an investigative journalist who faces a death threat from the Camorra after writing a best-selling book Gomorrah on the group, also made a surprise appearance at the protest.