Background The dynamics of coronavirus disease 2019 (COVID-19) seroconversion of hospital employees are understudied. Our aim was to measure the proportion of seroconverted employees and evaluate risk factors for seroconversion. Methods This prospective cohort study recruited Geneva University Hospitals employees sampled 3 times every 3 weeks from March 30 to June 12, 2020. We measured the proportion of seroconverted employees, and determined prevalence ratios of risk factors for seroconversion using mixed-effects multivariable Poisson regression models. Findings Overall, 3’421 participants (29% of all employees) were included with 92% follow-up. The proportion of seroconverted employees increased from 4·4% [95%CI, 3·7-5·1] at baseline to 8·5% [95%CI, 7·6-9·5] at the last visit. The proportions of seroconverted employees working in COVID-19 (32·3%) and non COVID-19 geriatrics and rehabilitation (G&R) wards (12·3%) were higher compared to office workers (4·9%) at the last visit. Only nursing assistants had a significantly higher risk of seroconversion when compared to office workers (11·7% vs 4·9%, p = 0·006). Significant risk factors for seroconversion included use of public transportation (adjusted prevalence ratio, 1·59 [95%CI 1·25-2·03]), community exposure to SARS-CoV-2 (2·80 [95%CI 2·22-3·54]), working in a ward with a nosocomial COVID outbreak (2·93 [95%CI 2·27-3·79]), and working in COVID-19 (3·47 [95%CI 2·45-4·91]) or non-COVID-19 G&R wards (1·96 [95%CI 1·46-2·63]). An association was observed between reported use of respirators and lower risk of seroconversion (0·73 [95%CI 0·55-0·96]). Interpretation Additional efforts are needed to protect employees in elderly care wards. There is an urgent need for randomized trials on the protective effect of respirators.