Healthcare workers are considered at higher risk for mumps infection than the general population. Since 2017, the national immunization plan recommended the administration of a dose of measles–mumps–rubella (MMR) vaccine to the healthcare operators who are unable to demonstrate a complete vaccination history or that are seronegative for at least one of the three agents. Regarding mumps infection, based on actual concerns regarding the loss of protection over the years after vaccination, the Advisory Committee on Immunization Practices (ACIP) recommended to administer a third dose of vaccine to operators previously vaccinated with two doses of MMR vaccine who belong to a group at increased risk of mumps infection in the event of an epidemic. This guideline, however, is not currently followed in Italy, resulting in a potential risk for vaccinated operators to become unprotected from mumps over the years. The aim of our study is to evaluate the persistence of a protective antibody level for mumps among medical students vaccinated during infancy or adolescence, at the start of their hospital internship. We retrospectively evaluated mumps-specific IgG levels in a group of medical students, in the period from 1 January to 31 December 2020. We evaluated the persistence of the detectable level of mumps-specific antibodies in relation to their vaccinal status, gender and time elapsed from vaccination. We found that 17.4% (65 subjects) of our sample were seronegative for mumps. The univariate analysis showed a significant difference in serological protection between male and female gender (77.0% vs. 86.2%; p < 0.05 with chi2 test) and between age classes (86.5% vs. 76.4%; p < 0.05 for subjects aged 18–23 years and over 23 years, respectively). Female gender was significantly related to higher serological protection even after adjusting for age classes and number of vaccine doses administered in a multivariate analysis model. Our study shows a substantial percentage of subjects lacking a protective mumps titer among medical students who were vaccinated in childhood. Given the higher risk of infection among those subjects, routine pre-employment screening should be performed among those operators regardless of their vaccination history and a third dose of MMR should be offered to unprotected students.