Background: Italy is considered an area with very low HAV (hepatitis A virus) endemicity. Currently in Italy the anti-HAV vaccine is recommended only for specific risk groups and there is no universal vaccination program. The aim of this study was to assess the level of immunity against hepatitis A in a sample of children and adolescents from the province of Florence. Methods: A total of 165 sera were collected from subjects aged 1 to 18 years, proportionally selected according to the general population size and stratified by age and sex. A qualitative evaluation of anti-HAV antibodies was performed using the enzyme-linked immunosorbent assay (ELISA). Anamnestic and vaccination status data were also collected. Results: Our study showed a hepatitis A seroprevalence of 9.1% in the enrolled population. A statistically significant difference in the prevalence of anti-HAV was found between Italian and non-Italian subjects. About half of the population having anti-HAV antibodies was reported to be vaccinated, and no cases of hepatitis A were found. Conclusions: The data from our study confirmed Tuscany as an area with low HAV endemicity and showed that hepatitis A seroprevalence is significantly higher in foreign children and adolescents. The presence of more seropositive subjects than those vaccinated was probably due to a natural immunization achieved through a subclinical infection and/or to underreporting of the surveillance systems.