The group called the regulation a "serious intrusion" on the ability of families to make their own health care decisions.
"While we understand the frustration of public health officials who have been unable to convince parents to willingly vaccinate their children against influenza, state intervention in the medical decisions of families must be limited and rare," ACLU policy associate Hillary Davis said at a hearing this month.
Under the draft rule, annual influenza vaccination would be mandatory for all children between 6 months and 59 months before entering public or private preschool or day care programs. Even students deemed exempt from the vaccination requirement for medical or religious reasons would be forced to stay home during an outbreak of flu.
Health Department spokesman Jim Palmer said Wednesday the agency is looking out for public health.
"It's difficult sometimes, if you decide not to vaccinate your children, but that can't be a threat to the greater community," he said.
Schools already require multiple vaccinations, including for mumps and measles, chickenpox and hepatitis B, among other diseases.
Davis said vaccines should be mandatory only when they reliably prevent the illness and when an unvaccinated child poses a high risk of infecting peers at school or day care. She noted the efficacy of the flu vaccine varies from year to year and that its protection is not lasting.
"It's an annual vaccine that doesn't always work," Davis said in an interview Wednesday. "It's just not appropriate for the Department of Health to be saying this is a required vaccination."
The proposed regulation says children exempt from the vaccination requirement shall be excluded from school for however long the health department decides based on a case-by-case analysis of public health risk. Davis said that could mean some unvaccinated children are kept home for weeks.
Workers in preschools and day care programs would also be required to be vaccinated against flu under the proposed rule.
The health department in 2012 made flu vaccinations for health care workers mandatory. Those who do not get the shot, including for medical reasons, are required to wear a surgical mask during periods when the state health director deems flu to be "widespread."
A union representing health care workers challenged the policy in court, saying no medical evidence exists that vaccinating workers protects patients or that masks inhibit the transmission of flu. The lawsuit was later withdrawn.
The health department has also proposed mandating the human papillomavirus vaccine series beginning with students entering seventh grade. The vaccine protects against cervical cancer and genital warts; the virus is passed through sexual contact.