Background Healthcare workers (HCWs) faced substantial risk of infection during the COVID-19 outbreak. This study aims to determine the prevalence of anti-SARS-CoV-2 antibodies in a cross-sectional sample of HCWs as well as risk factors associated with exposure to SARS-CoV-2. Methods The study was conducted between March and May 2021 at the American University of Beirut Medical Center (AUBMC), a tertiary hospital located in Lebanon. Socio-demographic and clinical data, as well as data on exposure, PCR results, PPE adherence, and vaccination status, were collected using an online questionnaire. Sera were also collected to determine seropositivity using commercially available enzyme-linked immunoassay (ELISA) targeting the spike (S) and the nucleocapsid proteins (NCP) of SARS-CoV-2. Findings Among 92 recruited HCWs, 72.3% received PPE training, more than 70% were adherent to using appropriate PPEs, and around 80% were vaccinated. Nurses in this study population were at higher risk of exposure compared to medical doctors, technicians, and other HCWs. Among the HCWs who performed a PCR test, 28.6% were infected with SARs-CoV-2 with workplace exposure not associated with COVID-19 infection. All vaccinated HCWs were seropositive for anti-S IgG with high titer (≥384 BAU/mL), with a significantly higher median anti-S IgG titer compared to unvaccinated HCWs with previous infection (384 vs. 140.1 BAU/mL; p = .0043). Conclusions Our study highlights the importance of implementing strict infection control policies among HCWs and deploying an effective COVID-19 vaccination strategy. More studies are needed in Lebanon to assess risk factors of SARS-CoV-2 exposure in the workplace.