Italy's so-called Lorenzin law, named after the former health minister who introduced it, states children must receive a set of mandatory injections before attending school.
Children up to the age of six years will be excluded from nursery and kindergarten without proof of vaccination under the new rules.
The two parties that make up Italy's government - The League and the Five Star Movement - had criticised the policy of compulsory vaccinations, both before and after they came to power last summer. These vaccines include polio, measles, mumps, rubella and chickenpox.
The fresh law took place amid an increase in measles cases, however, Italian officials declared that vaccination levels have been better since it was offered.
In Bologna, 300 children were suspended from school, according to Italian media reports, while 37 were turned away in the northern Veneto region.
"Now everyone has had time to catch up", Health Minister Giulia Grillo told La Repubblica. Those people believe that vaccines are risky and, depending on what misinformation they have absorbed, can cause everything from autism and attention deficit disorder to "vaccine overload", a made-up condition that is not an actual medical term.
"Italy is part of a global trend of distrust in mediators-doctors and scientists-who can interpret and explain data", La Sapienza University of Rome history of medicine and bioethics teacher Andrea Grignolio told CNN in 2018.