Background: The Work Ability Index (WAI) measures how well employees' abilities match their job demands. This study assessed the WAI among health workers and explored how age, gender, and job roles affected it. The research was conducted in a central Italian hospital, with a focus on health workers undergoing health surveillance between September 2020 and April 2021. Methods: Data were collected using validated questionnaires that assessed the WAI and risk factors for metabolic syndrome among participants. Demographic information, including age, gender, and occupation, was also obtained. The study involved 1847 health workers, with an average age of 43 years, predominantly women (67.6%). Occupational categories included administrative staff, nurses/healthcare workers (HCWs)/midwives, physicians, and healthcare technicians. Statistical analyses, such as t-tests, ANOVA, and chi-squared tests, were performed to explore the relationships between WAI scores and demographic/occupational variables. Results: The study suggested a relationship between WAI scores and gender, age, and occupation. Men workers exhibited higher mean WAI scores than women workers, while older workers (>55 years) had lower WAI scores compared with their younger counterparts. WAI scores varied by job role, with physicians scoring the highest. Conclusions: The findings suggested that demographic and occupational factors were associated with variations in work ability among health workers. These findings can help improve workforce management, occupational health, and research on aging workers. However, it is important to acknowledge the limitations of this study. Given its cross-sectional design, causal inferences cannot be established, and further longitudinal research is needed to confirm these findings and explore potential causal relationships.