We assessed the seroepidemiology of SARS-CoV-2 infection and the incidence of coronavirus disease 2019 (COVID-19) before and during the rollout of COVID-19 vaccines, in a prospective observational cohort study on healthcare workers (HCWs) in a large tertiary hospital in Mainz, Germany. Antibody status was assessed during six visits between September 2020 and February 2022. Self-reported symptoms were collected using a smartphone application; symptomatic HCWs were tested using real-time polymerase chain reaction (RT-PCR) assays for SARS-CoV-2. Rates of virologically confirmed and severe COVID-19 were estimated using the U.S. Food and Drug Administration (FDA) and Coalition for Epidemic Preparedness Innovations (CEPI) case definitions, respectively, and were contrasted to background community transmission and circulating SARS-CoV-2 variants. A total of 3665 HCWs were enrolled (mean follow-up time: 18 months); 97 met the FDA definition of virologically confirmed COVID-19 (incidence rate (IR) 2.3/1000 person-months (PMs), one severe case). Most cases reported ≥2 symptoms, commonly, cough and anosmia or ageusia. Overall, 263 individuals seroconverted (IR 6.6/1000 PMs—2.9 times the estimated IR of COVID-19), indicating many cases were missed, either due to asymptomatic infections or to an atypical presentation of symptoms. A triphasic trend in anti-SARS-CoV-2 seroprevalence and seroconversion was observed, with an initial increase following the rollout of COVID-19 vaccines, a two-fold decline six months later, and finally a six-fold increase by the end of the study when Omicron was the dominant circulating variant. Despite the increase in infection rates at the end of the study due to the circulation of the Omicron variant, the infection and disease rates observed were lower than the published estimates in HCWs and rates in the general local population. Preferential vaccination of HCWs and the strict monitoring program for SARS-CoV-2 infection are the most likely reasons for the successful control of COVID-19 in this high-risk population.