The active immunization of health care workers (HCWs) is a crucial measure to avoid nosocomial infection; nevertheless, vaccine coverage (VC) among health personnel in Italy is unsatisfactory. To improve VC in the healthcare set, the Hygiene and Occupational Medicine departments of Bari Policlinico General University Hospital applied a specific program. The operative procedure demands that in the context of the occupational medical examination, all workers are evaluated for susceptibility to vaccine-preventable diseases (VDPs), with immunization prophylaxis offered to those determined to be susceptible. This study analyzed data from workers who attended the biological risk assessment protocol from December 2017 to October 2021 (n = 1477), who were evaluated for the immune status for measles, mumps, rubella, and varicella. Among the enrolled subjects, non-protective antibody titers were higher for measles and mumps (13%), followed by rubella (11%) and varicella (8%). Appropriate vaccinations were offered to all susceptible HCWs, and HCWs were re-tested one month after immunization. The seroconversion rate after the administration of one or more booster dose(s) was over 80%. Overall, 2.5% of the subjects refused the offered vaccine(s); the main determinant of immunization compliance was younger age (aOR = 0.86; 95%CI = 0.80–0.92). Especially during the COVID-19 pandemic, VPDs may still present a hazard in nosocomial environment. Our experience suggests that, despite hospital procedures and dedicated human assets, satisfactory VC cannot be reached without the provision of federal regulations. Nevertheless, public health policymakers have to improve the promotion of vaccine prophylaxis and education to reach higher VC.