Objective: The risk of reinfection with SARS-CoV-2 has been rapidly increased with the circulation of concerns about variants. So, the aim of our study was to evaluate the factors that increase the risk of this reinfection in healthcare workers compared to those who have never been positive and those who have had only one positivity. Methods: A case-control study was carried out at the Teaching Hospital Policlinico Umberto I in Rome, Sapienza University of Rome, in the period between 6 March 2020 and 3 June 2022. Cases are healthcare workers who have developed a reinfection with the SARS-CoV-2 virus, while controls were either healthcare workers who tested positive once or those who have never tested positive for SARS-CoV-2. Results: 134 cases and 267 controls were recruited. Female gender is associated with a higher odds of developing reinfection (OR: 2.42; 95% CI: 1.38–4.25). Moreover, moderate or high alcohol consumption is associated with higher odds of reinfection (OR: 1.49; 95% CI: 1.19–1.87). Diabetes is also associated with higher odds of reinfection (OR: 3.45; 95% CI: 1.41–8.46). Finally, subjects with increased red blood cell counts have higher odds of reinfection (OR: 1.69; 95% CI: 1.21–2.25). Conclusion: From the prevention point of view, these findings indicate that particular attention should be paid to subjects with diabetes mellitus, women and alcoholic drinkers. These results could also suggest that contact tracing represents a fundamental approach model against the SARS-CoV-2 pandemic, together with the health information of participants.