The law was implemented as a response to a recent spike in measles outbreaks.
This stance follows months of debate over compulsory vaccination, both in Italy and across the world.
If a child under age 6 has not been vaccinated, they will not able to attend kindergartens or schools.
After the notorious study by Dr. Andrew Wakefield that linked measles vaccine to autism, there has been rise in anti-vaccination sentiments among parents.
"Italy's measles vaccine coverage was par with Namibia, lower than Ghana", San Raffaele University in Milan microbiology and virology professor Roberto Burioni told CNN past year.
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The new rules came into force on Tuesday and are part of the so-called "Lorenzin law", named after former Italian Health Minister Beatrice Lorenzin.
For years, confusion about vaccines has reigned in Italy. Italy's health minister, Giulia Grillo told La Repubblica newspaper that people were given ample time to get their ward's vaccinated and update the immunization records. Children between 6 and 16 can not be flat-out banned from attending school, but parents will face fines up to $560 for sending their unvaccinated kids to class.
Regional authorities are taking care of the situation through different ways, report Italian media.
In Bologna, the local authority has sent letters of suspension to the parents of some 300 children, and a total of 5,000 children do not have their vaccine documentation up to date. Such a low rate adversely affects herd immunity that prevents the spread of infection.
Newly released figures show that Italy is nearing, and in some regions has already reached, a national immunization rate of 95 per cent, the World Health Organization's target.
In British Columbia, which is now experiencing a small measles outbreak, parents started a petition calling for mandatory vaccinations to attend school.