Introduction: In Europe, there is still suboptimal tetanus, diphtheria, and acellular pertussis (Tdap) booster coverage. This study aimed to assess coverage status, knowledge, and attitude on Tdap vaccination in healthcare workers (HcWs) of the University Hospital “Federico II” in Naples, Southern Italy, in 2022, to improve current vaccination strategies.

Methods: A cross-sectional study was conducted using a validated anonymous questionnaire. Knowledge and attitude were measured as scores. Multivariable logistic and linear regression models were employed to identify correlates of Tdap booster and knowledge and attitude toward the vaccination, as appropriate. Models were controlled for age, sex, profession, department, and job seniority.

Results: A total of 206 questionnaires were administered among HcWs, and 143 (69.4%) were medical doctors. In total, 71 (34.47%) HcWs received the Tdap booster. Those who have worked 5–9 years at the hospital had a 78% lower likelihood of being vaccinated with the Tdap booster (5–9 years—OR: 0.22, CI: 0.06 | 0.85) as compared with newly hired HcWs. No differences in the average knowledge score were found. Other healthcare workers had a lower attitude as compared to medical doctors (Other—Coef. −2.15; CI: −4.14 | −0.15) and, as compared with those who worked in a clinical department, those who worked in a diagnostic–therapeutic department or medical management had 3.1 and 2.0 lower attitude scores, on average, respectively (diagnostic–therapeutic—Coef. −3.12, CI: −5.13 | −1.12; public health—Coef. −1.98, CI: −3.41 | −0.56).

Discussion: The study findings support the necessity to implement public health strategies and improve knowledge and attitude toward vaccinations and specifically highlight the importance of Tdap booster every 10 years as a prevention tool to protect high-risk populations.