The efficacy of the first schedule of COVID-19 mRNA vaccines has decreased after the surge of the Delta variant, posing the need to administer a booster dose to enhance the neutralising immune response. This study aims at evaluating the duration of protection given by the booster dose of Pfizer–BioNTech BNT162b2 mRNA vaccine in healthcare workers (HCWs) of a large teaching hospital in Rome and to analyse the factors associated with post-booster vaccination infections. Data about vaccinations of HCWs with the BNT162b2 vaccine and nasal swabs positive for SARS-CoV-2 were extracted from the digital archives of the hospital from 27 September 2021 to 31 May 2022. In total, 5770 HCWs were observed. The cumulative risk of becoming infected by SARS-CoV-2 increased with time (2.5% at 4 weeks, 17% at 12 weeks and 40% at 24 weeks) and was significantly higher for females, younger classes of patients and for those who had developed a hybrid immunity (natural infection plus one dose, namely the primary schedule, added to the booster dose) compared to those who had completed the three doses. This study describes the duration and the determinants of the protection against infections after the booster dose of COVID-19 vaccine, highlighting the need for continuous monitoring of vaccine-induced immunogenicity.