Under a new law, children under six will be legally turned away from preschool, which is known as "Asilo" in Italy.
Under the law, children over six can not be banned from school but their parents will be fined up to €500 (£425).
"Now everyone has had time to catch up", Health Minister Giulia Grillo told La Repubblica.
Some children were denied entry to schools on Tuesday but the numbers appeared to be modest.
Children who are unable to get vaccinated due to medical reasons are exempt from the requirement. It's unclear how many children will face suspensions from schools nationwide.
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.
They include vaccinations for chickenpox, polio, measles, mumps, and rubella. Officials in Bologna said that some 5,000 children in the city do not have proper documentation saying they have been vaccinated. ". No vaccine, no school".
The goal of the law, according to a government website, is to fight the gradual decline in Italy's vaccination rates. 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.
The law was passed in order to tackle an outbreak of measles after some 5000 cases were reported in 2017.