To combat the COVID-19 pandemic, a mass vaccination campaign was initiated in Italy on December 27, 2020. The vaccine available to immunize Italian healthcare workers (HCWs) was the BNT162b2 mRNA COVID-19 vaccine (Comirnaty). This study evaluated the effectiveness of the vaccine against documented SARS-CoV-2 infection and symptomatic diseases in the medium- to long-term. HCWs at Bari Policlinico University-Hospital (Italy) who completed the vaccination schedule were matched with HCWs who had refused vaccination; the two groups were followed-up for 5 months (January–May 2021). Vaccine effectiveness (VE) against infection was 97.7% (95.4–99.0%) at 14–34 days after the first dose, and 94.8% (87.0–97.8%), 83.0% (65.0–92.0%), and 81.0% (42.0–94.0%) at 14–41, 42–69, and >69 days, respectively, after the second dose. The estimated VE for documented symptomatic disease was 99.2% (96.4–99.8%) at 14–34 days after the first dose and 97.2% (90.3–99.2%), 85.0% (63.0–94.2%), and 88.0% (42.0–97.6%) at 14–41, 42–69, and >69 days, respectively, after the second dose. Efforts to increase vaccination rates should be strengthened, including mandatory vaccination for HCWs and greater incentives to increase vaccine acceptance by the general population.