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Background: Understanding the factors affecting humoral immune response to COVID-19 vaccines among healthcare workers (HCWs) is essential to predict their level of protection. Vaccination elicits antibodies against SARS-CoV-2 spike protein (anti-S). Aim: To investigate the factors associated with the presence of SARS-CoV-2 anti-S antibodies among vaccinated HCWs. Methods: This cross-sectional study included 143 vaccinated HCWs, with or without a history of previous COVID-19 infection (clinically, radiologically, or by laboratory results) from different departments. Socio-demographic, clinical, as well as vaccine-related data, were recorded. Serum samples were collected and tested for SARS-CoV-2 spike antibodies. Results: Vaccination provoked an immunogenic response, where the overall anti-S positivity was 83.9% (95% CI: 77.8–90.0%). The response was not affected either by the age or gender of HCWs. Out of the 143 HCWs, 46 (32.1%; 95% CI: 24.4–39.9%) reported a previous history of COVID-19 infection, and seropositivity was significantly higher among them (p = 0.002), and it was associated with the frequency of infection (p = 0.044) and duration since diagnosis of COVID-19 infection (p = 0.065). They had higher median anti-S titers (111.8 RU/mL) than those without infection (39.8 RU/mL). Higher seropositivity was observed with Oxford/AstraZeneca vaccine (AZD1222) (88.9%; 95% CI: 83.1–95.0%) than Sinopharm (BBIBP-CorV) (67.7%; 95% CI: 50.3–85.2%), and with receiving two doses of vaccine (92.3%; 95% CI: 87.1–97.5%). Conclusions: Antibody positivity was significantly affected by the previous history of COVID-19 infection, type of vaccine, the number of doses received, and duration since vaccination.