Under the "Lorenzin law", parents had until March 10, 2019, to provide documentation showing their child had been inoculated with a number of vaccinations, as reported by RAI News, Italy's national public broadcaster.
Children under 6 years old will be banned from attending nursery and kindergarten if parents can not provide proof of vaccination.
The legislation follows months of debate over compulsory vaccination and came amid a surge in measles cases.
But up until Tuesday, a temporary measure meant students could remain in school as long as their parents said they were vaccinated.
So far, it has been reported that in Bologna, authorities have sent suspension letters to parents over more than 300 children, and a total of 5,000 kids do not have up-to-date vaccination documents.
"Italy's measles vaccine coverage was par with Namibia, lower than Ghana", San Raffaele University microbiology and virology professor Roberto Burioni told CNN past year.
"Now everyone has had time to catch up", Health Minister Giulia Grillo told La Repubblica. ". No vaccine, no school".
The required vaccinations are polio, diphtheria, tetanus, hepatitis B, pertussis, measles, rubella, mumps, chicken pox and Haemophilus influenzae type B.
Regional authorities are taking care of the situation through different ways, report Italian media.
The BBC wrote that the law was passed to bolster flagging Italian vaccination rates, which is in part due to a growing movement of anti-vaccination activists (widely known as antiaxxers). "With the advent of the Internet, people have the illusion they can access and read data by themselves, removing the need for technical and scientific knowledge".
The law was implemented as a response to a recent spike in measles outbreaks.