Under the country's so-called Lorenzin law - named after the former health minister who introduced it - children will now be required to receive a range of immunizations in order to attend school.
According to the BBC, the local authority in Bologna has already sent letters of suspension to the parents of approximately 300 kindergarten children and around 5,000 don't have up-to-date vaccination documentation.
Under the law, children over six can not be banned from school but their parents will be fined up to €500 (£425).
Following months of fiery debate - and measles outbreaks - a new law banning unvaccinated children from Italy's classrooms has come into effect.
The city of Bologna reportedly has at least 300 children who now do not comply with the vaccination requirements and are at risk of suspension from school.
"Italy's measles vaccine coverage was par with Namibia, lower than Ghana", San Raffaele University microbiology and virology professor Roberto Burioni told CNN a year ago.
"Now everyone has had time to catch up", Health Minister Giulia Grillo told Italian newspaper La Repubblica, the BBC reported. However, the B.C. government has said that it is only planning to require mandatory reporting of vaccination status in the upcoming school year. The BBC added that Italian media reported regional authorities are "handling the situation in a number of different ways", with no notices of suspension reported in some areas and grace periods allowed in others.